Temperature Sensitive Mutants in Fission Yeast Marinova.pdfSingle Site Suppressors of a Fission Yeast Temperature-Sensitive Mutant in cdc48 Identified by Whole Genome Sequencing Irina

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F A C U L T Y O F S C I E N C E

U N I V E R S I T Y O F C O P E N H A G E N

D E N M A R K

PhD Thesis

Irina N. Marinova

Suppressor Analysis of CRL4Cdt2 Defective

and cdc48-353 Temperature Sensitive Mutants

in Fission Yeast

Academic advisors:

Professor Olaf Nielsen

Associate Professor Rasmus Hartmann-Petersen

Submitted: 24/08/16

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Suppressor Analysis of CRL4Cdt2

Defective and cdc48-353 Temperature

Sensitive Mutants in Fission Yeast

Irina Nikolaeva Marinova

PhD thesis

This thesis has been submitted to the PhD School of Science at the University

of Copenhagen, Denmark in August 2016

DENMARK

THE PHD SCHOOL OF SCIENCE

FACULTY OF SCIENCE

UNIVERSITY OF COPENHAGEN

DENMARK

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Preface

The work presented in this thesis was carried out in the Cell Cycle & Genome Stability

Lab, Department of Biology at the University of Copenhagen under the supervision of

Professor Olaf Nielsen and Associate Professor Rasmus Hartmann-Petersen between March

2013 and May 2016.

The thesis covers two main areas presented in two chapters.

Chapter 1 provides background on the cellular functions of the CRL4Cdt2

E3 ubiquitin

ligase and its target Spd1. The chapter presents unpublished work on the characterization of

spd1 mutants isolated as spontaneous suppressors of the growth defects exhibited by

CRL4Cdt2

-deficient fission yeast cells. Some of the experiments presented therein were

performed by others than myself, and their contribution is mentioned in the chapter.

Chapter 2 describes the identification of spontaneous suppressors of the growth defects

exhibited by the temperature-sensitive cdc48-353 fission yeast mutant using whole genome

sequencing approach. The chapter is based on the findings described in Paper I.

Paper I

Single Site Suppressors of a Fission Yeast Temperature-Sensitive Mutant in cdc48

Identified by Whole Genome Sequencing

Irina N. Marinova, Jacob Engelbrecht, Adrian Ewald, Lasse L. Langholm, Christian

Holmberg, Birthe B. Kragelund, Colin Gordon, Olaf Nielsen, Rasmus Hartmann-

Petersen

PLoS ONE 2015, doi:10.1371/journal.pone.0117779

As far as I am aware, the work described here is original, and has not been submitted

for any other degree.

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Acknowledgements

First of all, I would like to express my extreme gratitude to my supervisors Olaf Nielsen

and Rasmsus Hartmann-Petersen for their commitment to the work, excellent guidance and

constructive criticism. They have created an inspiring research environment and have

always been open for discussions. Their support has been an invaluable source of inspiration

throughout my studies.

I especially want to thank Christian Holmberg for his constant support, help with

experimental design, and all the fun we had while working together.

I also wish to thank the Faculty of Science, University of Copenhagen for funding my

PhD project.

This thesis contains unpublished work performed by others than myself. I would like to

acknowledge and thank our collaborators Birthe Kragelund and the colleagues from the

SBiN lab at the University of Copenhagen, who have been extremely helpful, positive and

willing to share their expertise. I highly appreciate the professionalism of Jacob Engelbrecht

and Adrian Ewald who have contributed substantially to this work. They have executed the

bioinformatical analysis of the data and Adrian Ewald has also performed key experiments

used in this thesis. I also thank Lasse Langholm for his help with the experimental work. I

would also like to thank Janni Petersen, Flinders University for performing experiments,

sharing strains and antibodies, and providing helpful discussions.

I would like to thank Professor Michael Lisby for providing access to his technical

equipment and instructions for the use of the Delta Vision fluorescent microscope.

I furthermore thank the The Danish National High-throughput Sequencing Centre for

their professional and flexible service.

I thank all my colleagues at the Cell Cycle & Genome Stability Lab, current and former

office mates for creating a wonderful work environment. I would particularly like to thank

Michaela Lederer and Karin Holm for their excellent technician assistance.

I would like to thank Olaf Nielsen for his help with the Danish Resumé and critical

comments on this thesis. Special thanks to Ieva Bagdonaite for her unconditional support,

proofreading and constructive criticism on the manuscript. I thank Miroslav Zashev for

helping me format this thesis.

Finally, I would like to thank my family and friends enormously for always being there

for me and encouraging me. I could not have done this without you!

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Contents

Preface ........................................................................................................................................ v

Acknowledgements .................................................................................................................. vii

Abbreviations ............................................................................................................................ xi

Summary ................................................................................................................................. xiii

Resumé ..................................................................................................................................... xv

Mutational Analysis of spd1 in Fission Yeast ......................................................................... 1

Introduction ............................................................................................................................ 3

DNA integrity checkpoints ........................................................................................... 3

Function and regulation of ribonucleotide reductase .................................................. 4

Ubiquitin-mediated protein degradation ..................................................................... 7

Structure and function of CRL4Cdt2

ligase....................................................................7

TORC2-Gad8 signaling in fission yeast ..................................................................... 14

Results.................................................................................................................................. 15

Screen for spontaneous suppressors of Δcdt2 growth defects ................................... 15

Amino acid residues V40 and S43 are important for the function of Spd1 ................ 16

The phosphomimetic S43D mutation does not affect the stability of Spd1 protein .... 22

Suc22R2

nuclear import function of Spd1 is not significantly affected by the V40G,

S43L and S43D mutations .......................................................................................... 23

Spd1 amino acid residues V40 and S43 are important for interaction with PCNA ... 25

Deletion of spd1 does not suppress the sensitivity of gad8 mutants to replication

stress and DNA damage ............................................................................................. 27

Overexpression of suc22 affects the interaction between Spd1 and PCNA ............... 28

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Discussion ............................................................................................................................ 29

Screen for Spontaneous Suppressors of the Fission Yeast Temperature Sensitive cdc48-

353 Mutant .............................................................................................................................. 33

Introduction .......................................................................................................................... 35

Results and Discussion ........................................................................................................ 39

Materials and Methods ............................................................................................................. 41

References ................................................................................................................................ 50

Paper I ..................................................................................................................................... 61

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Abbreviations

AAA-ATPase – ATPase associated with

various cellular activities

APC/C – anaphase-promoting

complex/cyclosome

Asn - aspargine

Asp – Aspartic acid

ASK1 – apoptosis signal-regulating kinase 1

ATM - ataxia-telangiectasia mutated

ATP – adenosine 5’-triphosphate

ATPase – adenosine 5’-triphosphatase

ATR - ataxia telangiectasia and Rad3-related

BiFC - bimolecular fluorescence

complementation

Cdc – cell division cycle

CDK – cyclin dependent kinase

CDT1 - chromatin licensing and DNA

replication factor 1

Cdt2 - Cdc-10-dependent transcript 2

Cds1 - Checking DNA Synthesis 1

Chk1/2 – checkpoint kinase 1/2

Cip1 – CDK-interacting protein 1

CMG complex - CDC45, MCM, GINS

complex

CRL - cullin RING E3 ligase

cs – cold-sensitive

C-terminal – carboxy terminal

Cul2/4 – cullin 2/4

DIC - differential interference contrast

Dif1 - damage-regulated import facilitator 1

DNA - deoxyribonucleic acid

dNTP - deoxyribonucleoside triphosphates

Ddb1 – DNA-damage binding 1

DSB – double strand break

Dun1 - DNA-damage uninducible 1

DWD - DDB1 binding WD40 proteins

ECL – enhanced chemiluminescence

ERAD – endoplasmic reticulum-associated

degradation

FAF1 – FAS-associated factor 1

Far1 – factor arrest 1

Gad8 – G1 arrest defective 8

GFP – green fluorescent protein

GINS complex – go-ichi-ni-san complex

HIF1α – hypoxia-inducible factor 1α

HRP – horse radish peroxidase

HU – hydroxyurea

IDP – intrinsically disordered protein

MCM - Minichromosome maintenance

protein

Mec1 - mitosis entry checkpoint 1

MBF – Mlu1 binding factor

Mik1 - mitosis inhibitory kinase 1

Mip1 - Mei2 interacting protein

MMS – methyl methane sulfonate

MSA – minimal sporulating agar

MSL – minimal sporulating liquid

mtDNA – mitochondrial DNA

nmt – no message in thiamine

Npl4 – nuclear protein localization 4

Nrm1 - negative regulator of MBF targets

nt - nucleotides

N-terminal – amino terminal

ORF – open reading frame

PBS-T – phosphate buffered saline tween 20

PCNA – proliferating cell nuclear antigen

PCR – polymerase chain reaction

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PIKK – phosphatidylinositol 3-kinase-related

kinase

PIP box – PCNA interacting protein box

Rad – radiation sensitive

Rbp1 – RNA-binding protein 1

Rbx1 - Ring-Box 1

ROC1/2 – RING of cullins

RING – really interesting new genes

RNA – ribonucleic acid

RNAi - RNA interference

RNR - ribonucleotide reductase

SDS-PAGE – sodium dodecyl sulphate

polyacrylamide gel electrophoresis

Ser – serine

Sin1 - Sapk interacting protein

SIM – SUMO interacting motif

Sml1 – suppressor of Mec1 lethality 1

Spd1 – S-phase delaying protein 1

S. cerevisiae – Saccharomyces cerevisiae

S. pombe – Schizosaccharomyces pombe

Ste20 – sterile 20

Suc22 – suppressor of the Cdc22

SUMO - small ubiquitin-like modifier

TCA – trichloroacetic acid

TLS - translesion synthesis

TOR – target of rapamycin

TORC1/2 – TOR complex 1/2

Trp – tryptophan

ts – temperature-sensitive

Tyr – tyrosine

UBX - ubiquitin regulatory X

UBXD7 - UBX domain-containing protein 7

UBXL - ubiquitin regulatory X-like

Ufd1 – ubiquitin fusion degradation 1

UV – ultraviolet

VHL - Von Hippel-Lindau

VCP - vasolin-containing protein

WAF1 – wild type p53-activated fragment 1

XPC - xeroderma pigmentosum

complementation group C

YEA – yeast extract agar

YEL – yeast extract liquid

YFP – yellow fluorescent protein

Yox1 - yeast homeobox

5-FOA - 5-fluoroorotic acid

Mutational Analysis of spd1 in Fission Yeast

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Summary

Part 1

CRL4Cdt2

E3 ligase is a key regulator of cellular proliferation and genome integrity, as

it promotes the degradation of proteins involved in cell cycle progression, DNA

replication and repair. In fission yeast the small intrinsically disordered protein Spd1 is

targeted for degradation upon entry into S-phase and following DNA damage via

CRL4Cdt2

-mediated ubiquitylation, which also requires interaction with proliferating cell

nuclear antigen (PCNA). Spd1 is a negative regulator of ribonucleotide reductase (RNR),

the activity of which is required for deoxyribonucleotide (dNTP) synthesis. CRL4Cdt2

mutants, which fail to degrade Spd1, have decreased dNTP pool and their viability is

dependent on constitutive activation of the DNA integrity checkpoint. However,

accumulation of Spd1 in RNR mutants with elevated dNTP levels still causes checkpoint

activation and dependency. Here, we show that Spd1 amino acid residues V40 and S43 are

important for its function as an inhibitor of DNA synthesis, as the spd1-V40G and spd1-

S43L mutants were identified as spontaneous suppressors of the defective phenotypes

exhibited by cells with abrogated CRL4Cdt2

pathway. We confirm that these mutations

alleviate the checkpoint dependency, the DNA damage sensitivity and the meiotic defects

associated with Spd1 accumulation. Further analysis showed that whereas the V40G and

S43L substitutions do not have a significant impact on Suc22R2

nuclear import function of

Spd1, they affect the interaction between Spd1 and PCNA. Our results provide evidence

that excess Spd1 causes replication stress and genome instability by inhibiting PCNA.

Furthermore, we examined the potential regulation of Spd1 function by Gad8-mediated

phosphorylation of residue S43.

Part 2

Cdc48/p97 is a ring-shaped homohexameric chaperone-like complex involved in

numerous cellular processes, including protein degradation, cell cycle control, DNA

repair, and vesicle fusion. The cdc48 gene is essential in fission yeast and mutations or

changes in Cdc48/p97 protein expression have been linked to neurological disorders and

cancer in humans. To gain further insight into the function of Cdc48/p97, we performed a

screen for pseudo revertants of the cdc48-353 temperature-sensitive fission yeast mutant.

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Altogether, we isolated 28 independent spontaneous cdc48-353 suppressors that had also

acquired a cold-sensitive phenotype. Using whole genome sequencing approach, we

showed that these suppressors were all second site intragenic cdc48 mutants. Mapping the

suppressor mutations on the Cdc48 structure revealed that whereas the original G338D

lesion was located near the central pore of the hexameric ring, the suppressor mutations

were positioned at the subunit-subunit and inter-domain boundaries. Furthermore, we

isolated a suppressor which had not acquired a cold-sensitive phenotype and carried an

extragenic frame shift mutation in the ufd1 gene, which encodes a known co-factor of

Cdc48. Collectively, our results provide evidence that the structural stability of the Cdc48-

353 hexamer is perturbed at the restrictive temperature, but stabilized in the suppressors.

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Resumé

Del 1

E3 ubiquitinligasen CRL4Cdt2

fremmer nedbrydningen af proteiner involveret i

cellecyklusprogression, DNA-replikation og reparation, og er således en vigtig regulator

af cellulær proliferation og genom integritet. I spaltegær medierer CRL4Cdt2

ubiquitylering

og efterfølgende nedbrydning af det lille ustrukturerede protein Spd1 ved initiering af S-

fase eller efter DNA beskadigelse, en proces som også kræver interaktion med PCNA.

Spd1 er en negativ regulator af enzymet ribonukleotidreduktase (RNR), hvis aktivitet er

nødvendig for deoxyribonukleotid (dNTP) syntese. I CRL4Cdt2

mutanter nedbrydes Spd1

ikke, og dNTP pools er derfor reducerede og cellernes levedygtighed afhænger af

konstitutiv aktivering af DNA-integritetscheckpointet. Akkumulering af Spd1 proteinet i

RNR mutanter med forhøjede dNTP niveauer forårsager imidlertid stadig checkpoint

aktivering og afhængighed. Her viser vi, at Spd1 aminosyreresterne V40 og S43 er vigtige

for Spd1s funktion som inhibitor af DNA-syntese. Mutanterne spd1-V40G og spd1-S43L

blev identificeret som spontane suppressorer af den vækstdefekt, der udvises af celler med

en inaktiveret CRL4Cdt2

pathway. Vi viser, at disse mutationer suppresserer checkpoint

afhængighed, følsomhed overfor DNA skader, samt den meiotiske defekt som er

forbundet med Spd1 akkumulering. Yderligere analyse viste, at mens V40G og S43L

substitutionerner ikke har en signifikant indvirkning på Spd1’s nukleare importfunktion af

Suc22R2

, så påvirker de samspillet mellem Spd1 og PCNA. Vores resultater dokumenterer,

at overskydende Spd1 forårsager replikationsstress og genom-ustabilitet ved at hæmme

PCNA. Desuden undersøgte vi en mulighed regulering af Spd1s funktion gennem Gad8-

medieret phosphorylering af rest S43.

Del 2

Cdc48/p97 er et ringformet homohexameric chaperone-lignende kompleks involveret

i talrige cellulære processer, herunder proteinnedbrydning, cellecykluskontrol, DNA-

reparation, og vesikel-fusion. Genet for Cdc48 er essentielt i spaltegær, og mutationer eller

ændringer i Cdc48/p97-proteinekspression er forbundet med neurologiske lidelser og

cancer hos mennesker. For at få yderligere indsigt i funktionen af Cdc48/p97, udførte vi

en screen for pseudo-revertanter af en cdc48-353 temperaturfølsom mutant i spaltegær.

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Alt i alt, isolerede vi 28 uafhængige spontane cdc48-353 suppressorer, der samtidigt havde

erhvervet en kulde-følsom fænotype. Ved hjælp af en whole genome sequencing tilgang,

viste vi, at disse suppressorer alle repræsenterede intrageniske cdc48 mutanter.

Kortlægning af suppressor-mutationer på Cdc48 strukturen afslørede, at mens den

oprindelige G338D læsion var placeret nær den centrale pore af hexamere ringen, var

suppressor-mutationerne placeret ved subunit-subunit og inter-domænegrænserne.

Endvidere isolerede vi en enkelt suppressor som ikke havde erhvervet en kold-følsom

fænotype. Genom sekventering viste at denne havde rammeskiftmutation i ufd1 genet,

som koder for en velkendt co-faktor for Cdc48. Samlet, indikerer vores resultater, at den

strukturelle stabilitet af Cdc48-353 hexameren er kompromitteret ved den restriktive

temperatur, mens suppressor-mutationerne stabiliserer den igen.

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Chapter 1


Chapter objectives:

To investigate further the biological

function of Spd1-Suc22R2

and Spd1-

PCNA interactions, as well as to study

the possibility that Spd1 function is

regulated by phosphorylation of

amino acid residue S43


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Introduction

Fission yeast Spd1 is an intrinsically disordered protein, which negatively regulates

dNTP synthesis by inhibiting ribonucleotide reductase. Upon entry into S-phase or

following DNA damage, Spd1 is targeted for degradation via CRL4Cdt2

-mediated

ubiquitylation, which also requires interaction with DNA-loaded PCNA. Failure to

degrade Spd1, as in CRL4Cdt2

mutants, results in numerous cellular defects including

increased mutation rates, DNA damage sensitivity, inability to undergo premeiotic S-

phase, and constitutive checkpoint activation. Although the impact of Spd1 on genome

integrity has been extensively researched, its biological function is not fully understood.

The aim of this project was to provide further insight into the function and regulation of

Spd1-mediated protein interactions. In this study we identified two mutations in the spd1

gene, V40G and S43L, as spontaneous suppressors of the physiological defects displayed

by mutants with inactive CRL4Cdt2

E3 ubiquitin ligase. Detailed characterization of these

mutations revealed that their suppressive effect is most likely mediated through changes in

the interaction between Spd1 and PCNA. Furthermore, we provided insight that Spd1

function is possibly regulated by phosphorylation at residue S43, which is mediated by

Gad8 kinase. Hopefully, the findings presented herein will contribute to better our

understanding of Spd1 function and its impact on genome stability. Topics related to the

background of the project will be reviewed in the following sections. First, the DNA

integrity checkpoint as a mechanism for maintaining genome stability will be introduced,

followed by a review of the function and regulation of ribonucleotide reductase. Special

emphasis is put on the role of the CRL4Cdt2

E3 ubiquitin ligase and its targets Spd1 and

p21 in the regulation of cell cycle progression. Finally, the TORC2-Gad8 signaling

pathway and its possible functional intersection with CRL4Cdt2

ligase in maintaining

genome integrity will be touched upon.

DNA integrity checkpoints

Exposure of eukaryotic cells to genotoxic stress results in cell cycle arrest in order to

provide time for the damage to be repaired. The DNA damage and DNA replication

checkpoint mechanisms are responsible for monitoring the genome integrity by sensing


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DNA perturbations and transducing the signal to the cell cycle machinery (Weinert and

Hartwell, 1988; Hartwell and Weinert, 1989; Murakami and Nurse, 2000). DNA damage is

detected by sensor proteins which then trigger the signaling pathway that activates DNA

damage repair (Stracker et al., 2009). Detection of DNA damage activates the central

checkpoint transmitters ATR (Rad3 in S. pombe, Mec1 in S. cerevisiae) and ATM (Tel1

and S. pombe and S. cerevisiae), members of the evolutionary conserved subfamily of

phosphatidylinositol 3-kinases (PIKK), which further phosphorylate and stimulate the

downstream effector kinases CHK1 (Chk1 in S. pombe and S. cerevisiae) and CHK2

(Cds1 in S. pombe, Rad53 in S. cerevisiae). In yeast the Rad3ATR

is the major signal

transducing kinase (Alao and Sinnerhagen, 2008). In fission yeast Rad3ATR

is required for

the activation of both Cds1 and Chk1 downstream kinases (Walworth et al., 1993; Lindsay

et al., 1998). S. pombe Δrad3 mutants are defective in DNA integrity checkpoint activation

and loss of rad3 is lethal when DNA synthesis is perturbed by the presence of hydroxyurea

(HU) or other DNA damaging agents (Carr, 2002).

Cds1 is activated in response to replication stress and helps the cell to stabilize stalled

replication forks (Bahler, 2005), whereas Chk1 is activated in response to DNA damage in

late S/G2 and induces cell cycle arrest at the boundary of G2/M (Walworth et al., 1993;

Walworth and Bernards, 1996).

Function and regulation of ribonucleotide reductase

Precise DNA synthesis is essential for maintaining genome integrity and requires the

availability of balanced deoxyribonucleoside triphosphate (dNTP) pool (Chabes et al.,

2003; Holmberg et al., 2005). Generally, dNTPs can be synthesized through de novo and

salvage pathways. Failure to support proper dNTP concentrations promotes replication

fork collapse, occurrence of DNA breaks and higher risk of mutagenesis. In humans

unbalanced dNTP pools contribute to mitochondrial disorders, susceptibility to viral

infection, and cancer (Bester et al., 2011; Aye et al., 2015).

Ribonucleotide reductases (RNRs) are responsible for the de novo synthesis of dNTPs

in all organisms, as they catalyze the rate-limiting step of dNTP production by reducing

ribonucleoside diphosphates to their deoxy forms (Nordlund and Reichard, 2006). RNRs

are grouped in three classes based on the mechanism of free radical generation required for


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catalysis (Lundin et al., 2010). Eukaryotes use Class 1a RNRs which are aerobic enzymes

composed of multimers of two subunits – a larger subunit (R1, α) and a smaller subunit

(R2, β). In S. cerevisiae there are two genes encoding R1 (RNR1 and RNR3) and two genes

encoding R2 (RNR2 and RNR4) (Elledge and Davis, 1989 a; Elledge and Davis, 1990;

Huang and Elledge, 1997). In S. pombe, the R1 subunit, encoded by the cdc22 gene,

provides the catalytic activity of the enzyme, whereas the R2 subunit, encoded by the

suc22 gene, creates reducing power for R1 (Fernandez–Sarabia et al, 1993; Nordlund and

Reichard, 2006; Guarino et al., 2014).

RNR activity is strictly regulated at several levels throughout the cell cycle and its

critical function for proliferation and maintenance of genome stability is exploited in

chemotherapy of several types of cancer (Bonate et al., 2006; Shao et al., 2006).

Transcriptional regulation of RNR

In budding yeast transcription of the RNR1 gene is the major target for cell cycle

differential regulation upon S-phase entry, and expression of all RNR genes is strongly

promoted in response to DNA damage (Elledge and Davis, 1989 a, b; Elledge and Davis,

1990; Huang and Elledge, 1997; Huang et al., 1998). In fission yeast transcription of the

cdc22 gene is induced at G1/S transition and after DNA damage (Maqbool et al., 2003;

Gomez-Escoda et al., 2011), while the suc22 gene produces a shorter, constitutively

expressed transcript and a longer one induced by DNA damage (Harris et al., 1996).

Allosteric regulation

In addition to transcriptional regulation, RNR activity is modulated by allosteric

regulation. The R1 subunit contains two distinct effector-binding allosteric sites – the

specificity (S) site and the activity (A) site. The S site regulates the types of nucleotides to

be reduced, thus ensuring the supply of balanced dNTP ratios. The A site regulates the

overall RNR activity by binding dATP or ATP, which inhibits or stimulates the enzyme,

respectively (Nordlund and Reichard, 2006; Hofer et al., 2012). The precise stoichiometry

of the RNR complex is still not well characterized, but recent structural findings suggest

the formation of α6β2 oligomeric ring for the inactive and, possibly also the active

conformation of the enzyme (Fairman et al., 2011; Hofer et al., 2012). RNR can be locked


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in a constantly active state by substitution of the conserved Asp57 in the active site with

Asn, which disrupts the dATP-mediated allosteric feedback inhibition of the enzyme.

Mammalian cell lines and yeast bearing the D57N mutation have elevated dNTP pool and

increased mutation rate (Caras and Martin, 1988; Chabes et al., 2003, Fleck et al., 2013),

which demonstrates that maintaining adequate dNTP levels is crucial for genome integrity.

Regulation by small intrinsically disordered proteins

A further mechanism of controlling RNR activity employed only in yeast involves

small intrinsically disordered proteins (IDPs) that either bind directly to and restrict the

enzyme, or affect the subcellular localization of RNR subunits (Guarino et al., 2014).

Work in S. cerevisiae has demonstrated that the small Dif1 protein downregulates RNR by

binding to R2 and promoting its nuclear localization in non-S-phase cells (Lee et al., 2008;

Wu and Huang, 2008), whereas another small protein, Sml1, binds R1 and inhibits RNR

activity (Chabes et al., 1999). During replication or in response to DNA damage, both Dif1

and Sml1 are targeted for degradation by activation of the Mec1/Rad53/Dun1 checkpoint

pathway, thus allowing RNR activation and dNTP supply for DNA synthesis (Zhao et al.,

1998; Zhao et al., 2001; Lee et al., 2008). The MEC1 gene is essential for cellular viability

even in unperturbed cell cycle. Checkpoint-defective mutants fail to degrade Sml1 which

restricts dNTP synthesis during S-phase. Deletion of SML1 in mutants with inactive

Mec1/Rad53 pathway restores cell viability, as DNA replication is no longer disturbed

(Zhao et al., 2001).

In S. pombe, Spd1 is a functional orthologue of budding yeast Dif1 and Sml1 (Lee et

al., 2008), and regulates RNR activity by several mechanisms. As in S. cerevisiae,

functional RNR complexes are formed in the cytoplasm. The majority of Cdc22R1

is

constitutively cytoplasmic throughout the cell cycle, whereas outside S-phase, when RNR

activity is not required, Suc22R2

is localized mainly in the nucleus. During replication or

after DNA damage, Suc22R2

delocalizes to the cytoplasm where it interacts with Cdc22R1

to promote dNTP production (Liu et al., 2003; Holmberg et al., 2005). Suc22R2

nuclear

sequestration is dependent on the presence of Spd1, as deletion of the spd1 gene leads to

pan-cellular localization of Suc22R2

(Liu et al., 2003, Nestoras et al., 2010). Additionally,

Spd1 can inhibit RNR activity through binding to Cdc22R1

. Recent studies suggest that


7

Spd1 is able to interact with both RNR subunits and modulate the overall architecture of

the enzyme (Håkansson et al., 2006; Nestoras et al., 2010).

Ubiquitin-mediated protein degradation

Timely regulation of protein turnover is essential for maintaining cellular homeostasis.

Controlled proteolysis is achieved by the ubiquitin-proteasome system, where proteins

destined for destruction are tagged by covalent attachment of multiple ubiquitin molecules,

which targets them for degradation by the 26S proteasome (Havens and Walter, 2011).

Ubiquitin is a small (8 kDa) conserved protein which is conjugated to its targets by the

concerted action of three enzymes: (i) ubiquitin is first attached to an E1 ubiquitin-

activating enzyme; (ii) next, ubiquitin is transferred to an E2 ubiquitin-conjugating

enzyme; (iii) finally, an E3 ubiquitin ligase interacts with the substrate and the ubiquitin-

charged E2 enzyme, and mediates the transfer of ubiquitin to an internal lysine of the

substrate. In addition to ligating a single ubiquitin molecule to one lysine residue, or

multiple ubiquitin molecules to individual lysine residues of the substrate (called mono-

and multi-ubiquitylation, respectively), further ubiquitin moieties can be attached to the

first ubiquitin leading to the formation of polyubiquitin chain (polyubiquitylation). Mono-

and multi-ubiquitylation are protein modifications with important role in DNA synthesis

and repair, regulation of transcription, histone remodeling, etc., while polyubiquitylation

usually directs the protein for proteasomal degradation (Al-Hakim et al, 2010; Lipkowitz

and Weissman, 2011; Li and Jin, 2012).

Structure and function of CRL4Cdt2

ligase

Architecture of CRL4Cdt2

ligase

Cullin-RING E3 ligases (CRLs) comprise the largest subfamily of E3 ligases. CRLs

are modular ubiquitin ligases consisting of multiple subunits including (i) a scaffold

subunit from the conserved cullin family, (ii) a substrate recognition module, and (iii) a

small catalytic RING finger protein (ROC1 or ROC2), which interacts with an ubiquitin-

charged E2 enzyme (Figure 1) (Jackson and Xiong, 2009).

CRL4Cdt2

ligase contains the cullin scaffold Cul4 (Pcu4 in fission yeast) which binds

via its C-terminus to the RING finger protein Rbx1. The N-terminus of Cul4 associates


8

with the adaptor protein Ddb1, which can interact with multiple substrate receptors (or

DCAF - Ddb1-Cullin4 associated factors) (Petroski and Deshaies, 2005; He et al., 2006).

Cdt2 has been identified as a DCAF component of the Cul4-Ddb1 E3 ligase in fission

yeast and humans (Liu et al., 2005; Jin et al., 2006). Selective activity of the CRL4Cdt2

complex during replication and after damage is achieved by controlled expression of the

cdt2 gene throughout the cell cycle. During unperturbed S-phase, cdt2 transcription is

induced by the MBF transcription complex, while following DNA damage, cdt2 is induced

in a checkpoint-dependent manner by activation of the Rad3ATR

pathway (Liu et al., 2005).

Fig. 1. Structure and composition of Cullin-RING ubiquitin ligases. Cullin proteins (green) provide a

scaffold for the assembly of multi-subunit complexes that target specific substrates (light blue) for

degradation via ubiquitylation. Two paralogues (CUL4A/B) exist in mammalian cells (bottom, right). In

CRL4 ligases, the N-terminal domain of Cullin4 associates with the adaptor protein DDB1 (dark blue) which

interacts with the DWD motif (orange) from the substrate receptor (black). The C-terminal end of cullins

bind a small catalytic RING protein (ROC1 or ROC2, yellow) which interacts with an E2 ubiquitin

conjugating enzyme (red) that transfers ubiquitin (Ub) to the substrate. Cullins are activated by covalent

attachment of NEDD8 (Nd8, violet). From (Jackson and Xiong, 2009).


9

CRL4Cdt2

mediates ubiquitylation of substrates bound to DNA-loaded PCNA

Replication-coupled degradation of cell cycle proteins provides the cell with complete

and accurate genome duplication. The mechanism of replication-coupled proteolysis is

mediated by the CRL4Cdt2

E3 ligase. CRL4Cdt2

ligase ubiquitylates substrates bound to the

DNA-loaded fraction of PCNA (PCNADNA

) and PCNA is loaded on the chromatin during

DNA synthesis, which includes S-phase and DNA repair (Havens and Walter, 2011; Li

and Jin, 2012).

PCNA is a processivity factor for the replicative DNA polymerases and serves as a

molecular platform for the assembly of numerous partners involved in protein-protein and

protein-DNA interactions at the chromatin (Mailand and Bekker-Jensen, 2013). Proteins

that bind to PCNA contain a conserved motif (PIP box) which binds to a hydrophobic

pocket on the surface of PCNA (Moldovan et al., 2007). Furthermore, targets of the

CRL4Cdt2

ligase contain a PIP degron, which is a specialized PIP box comprising an

internal TD motif at positions 5 and 6 (Figure 2A). This motif confers high-affinity

binding to the PCNA allowing effective CRL4Cdt2

-assisted ubiquitylation (Havens and

Walter, 2009; Michishita et al., 2011). Moreover, mutational genetic analyses have

identified a basic amino acid four residues downstream of the PIP box (B+4) as an

essential element of the PIP degron, being crucial for the recruitment of CRL4Cdt2

to the

PCNA-substrate complex (Havens and Walter, 2009; Michishita et al., 2011, Guarino et

al., 2011).

Proteins which have evolved tight binding affinity to PCNA are suggested to be able

to compete out other PCNA interacting partners (Warbrick et al., 1997; Ducoux et al.,

2001). Therefore, clearing of substrates strongly bound to the PCNA might be critical

when other factors need access to the processivity factor to execute their function. In this

context, translesion synthesis (TLS) DNA polymerases are required for carrying out

replication across DNA lesions. TLS polymerases contain an ubiquitin-binding motif

which mediates their recruitment to PCNA by recognizing an ubiquitin moiety attached to

the processivity factor (Bertolin et al., 2015). Thereby CRL4Cdt2

has a regulative role in

TLS DNA synthesis, as it mediates the monoubiquitylation of PCNA in response to DNA

damage (Teary et al., 2010). In addition, it assists the switch between PCNA partners by


10

targeting for degradation proteins tightly associated with PCNA, thus triggering the

interaction with repair factors (Tsanov et al., 2014).

Interestingly, PIP degrons which confer strong PCNA interaction do not necessarily

ensure rapid degradation of the substrate. Recent study provides evidence that CRL4Cdt2

substrates are degraded in a strict sequential order based on the differences in the

efficiency with which PIP degrons recruit the adapter Cdt2 (Coleman et al., 2015). Thus

CRL4Cdt2

is a key player in molecular events programmed to take place in certain order,

which ensures correct S-phase progression and prevents genome disturbance.

Targets of the CRL4Cdt2

ligase

A number of proteins have been recognized as targets of the CRL4Cdt2

ligase in

humans and yeast including the replication licensing factor CDT1, the cyclin-dependent

kinase (CDK) inhibitor p21, the polymerase δ subunit p12, the histone methyl-transferase

SET8 and the RNR inhibitor Spd1 (Arias and Walter, 2005; Kim et al., 2008; Terai et al.,

2013; Zhang et al., 2013; Abbas, et al., 2010; Centore et al., 2010; Liu et al., 2003;

Holmberg et al., 2005).

Spd1

Spd1 was originally isolated as a multicopy inhibitor of cell cycle progression in S. pombe

(Woollard et al., 1996). Spd1 is a negative regulator of S-phase and elevated levels of

Spd1 cause cell cycle arrest in G1 and G2 phase, as well as checkpoint activation

(Woollard et al., 1996; Borgne and Nurse, 2000). No apparent sequence homology of

Spd1 has been identified in multicellular organisms. Spd1 is an IDP (Nestoras et al., 2010)

and its main biological function is to inhibit the activity of RNR. As mentioned earlier,

fission yeast Spd1 is a functional orthologue of S. cerevisiae Dif1 and Sml1, and in short

stretches it exhibits limited sequence similarity to both of them (Lee et al., 2008). Spd1

and Dif1 regulate R2 nuclear sequestration, and they contain a Hug domain, which is

required for interaction between Dif1 and R2 in budding yeast (Lee et al., 2008; Wu and

Huang, 2008). Genetic studies suggest that the Hug domain has similar function in Spd1

(Nestoras et al., 2010), and recently it has been shown that the N-terminal part of the Spd1

Hug domain comprises a PIP-degron required for interaction with PCNA (Figure 2B)

(Salguero et al., 2012). Similarly to Sml1, Spd1 can also bind and inhibit R1 (Håkansson


11

et al., 2006) and both proteins share a region of sequence similarity corresponding to the

Rnr1 binding domain of Sml1 (Zhao et al., 2000; Lee et al., 2008).

Spd1 is a key substrate of the CRL4Cdt2

ligase and as an inhibitor of DNA synthesis, it

is degraded during S-phase and DNA repair (Holmberg et al., 2005; Liu et al., 2003; Liu et

al., 2005; Bondar et al., 2004). Spd1 is recruited to the Cul4-Ddb1 complex through the

adaptor protein Cdt2, the expression levels of which are cell cycle regulated (Liu et al.,

2005). During S-phase, Spd1 degradation does not require activation of the Rad3ATR

pathway, whereas after DNA damage, Spd1 destruction becomes dependent on checkpoint

activation, corresponding to the fluctuations of cdt2 transcriptional induction (Liu et al.,

2005). However, high Cdt2 levels are not sufficient for promoting Spd1 proteolysis and

further requirement is the interaction between Spd1 and PCNADNA

(Salguero et al., 2012).

Fig. 2. (A) Consensus sequences of PIP box and PIP degron. Canonical PIP box residues (violet); PIP degron

specific residues (light blue); the TD motif at position 5 and 6 confers high affinity binding to PCNA. The basic

residue B at position +4 is required for recruiting CRL4Cdt2

to the DNA-loaded PCNA-substrate complex.

Numbers represent the positions of the residues in the PIP box. Residues downstream the PIP box are designated

with “+ “. (B) S. pombe Spd1protein. Top: Residues matching the PIP degron consensus sequence, are marked

with colour (violet and light blue, see A) as defined earlier (Salguero et al., 2012). Position of the residues in the

PIP box is denoted from 1 to +4. Q30 and S43 numbering indicates the position of the residues in the Spd1

protein. Bottom: S. pombe Spd1 protein (white bar) contains a Hug domain (dark blue box), encompassing amino

acid residues from 30 to 58. The Hug domain is required for interaction with R2. The PIP degron (red box)

required for binding to PCNA is located at the N-terminal end of the Hug domain. R1 binding domain (Lee et al.,

2008) is shown as an orange box. The position and range of boxes are relative and picture is out of scale. Adapted

from (Havens and Walter, 2011; Vejrup-Hansesn et al., 2014; Guarino et al., 2014; Salguero et al., 2012).


12

p21WAF1/Cip1

p21 (also known as WAF1 or Cip1) is a small intrinsically disordered protein (Yoon et

al., 2012), which was first established to act as a negative regulator of the cell cycle in

mammals (Harper et al., 1993). p21 mediates its biological activities mainly by binding to

and inhibiting the cyclin dependent kinases CDK1, CDK2, CDK4 and CDK6 (the main

kinases required for cell cycle progression in human), as well as through direct interaction

with PCNA, thus interfering with the process of DNA synthesis (Karimian et al., 2016;

Abbas and Dutta, 2009). p21 contains a C-terminal PCNA-binding domain comprising a

PIP degron and an N-terminal CDK-cyclin binding domain (Chen et al., 1995; Luo et al.,

1995). Through binding with high affinity to PCNA, p21 efficiently prevents the access for

other PCNA interacting partners (like DNA polymerase δ), thus directly inhibiting DNA

synthesis (Waga et al., 1994; Cazzalini et al., 2003). p21 expression is cell cycle regulated

where its levels peak in G1 and G2, and drop significantly in S-phase and after UV-

induced DNA damage. At G1/S transition and during S-phase, p21 is targeted for

degradation by the SCFSkp2

ubiquitin ligase (Bornstein et al., 2003; Wang et al., 2005) and

at G2/M transition p21 is ubiquitylated by the APC/CCdc20

ubiquitin ligase (Amador et al.,

2007). Recent studies revealed that CRL4Cdt2

ligase promotes destruction of PCNA-bound

fraction of p21 during S-phase and after DNA damage (Abbas et al., 2008; Kim et al.,

2008, Nishitani et al., 2008). Interestingly, the ubiquitylation and degradation of p21 after

UV irradiation has been shown to be enhanced by ATR-dependent phosphorylation of p21

at Ser114, suggesting that specific post-translational modifications might be required for

CRL4Cdt2

-assisted substrate destruction (Abbas et al., 2008; Lee et al., 2007).

While the function of p21 as a cell cycle inhibitor is related to its nuclear localization,

its stabilization and accumulation in the cytoplasm is associated with inhibition of

programmed cell death, thus promoting cell survival (Karimian et al., 2016). Akt1-

mediated phosphorylation of p21 within its PIP box at Thr145 and Ser146 disrupts the

binding between p21 and PCNA, thus triggering p21 cytoplasmic localization (Scott et al.,

2000; Rössig et al., 2001; Li et al., 2002). Outside the nucleus, phosphorylated p21

stimulates cell survival by suppressing apoptosis through inhibition of apoptosis-related

factors like ASK1, by releasing PCNA and Cdk2 inhibition, and by mediating the

formation and nuclear import of active CDK4/6-cyclin D complexes (Liang and


13

Slingerland, 2003; Abbas and Dutta, 2009; Yoon et al., 2012; Karimian et al., 2016).

Furthermore, it is considered that the ratio of relative abundance of p21 compared to Cdk-

cyclin complexes defines whether the Cdk-complex is activated or inhibited, where the

higher levels of p21 presumably stimulate the activity of Cdk2 and Cdk4 complexes with

cyclins (Zhang et al., 1998; LaBaer et al., 1997). Thus, p21 has a remarkable set of

interaction partners, which is a characteristic feature for IDPs, and fine tuning of p21

levels throughout the cell cycle is critical for the cell, as dysregulation of its abundancy is

implicated in various types of tumors (Abbas and Dutta, 2009; Yoon et al., 2009).

Cellular defects associated with CRL4Cdt2

inactivation

Fission yeast CRL4Cdt2

mutants, which fail to degrade Spd1, exhibit a number of

defective phenotypes including low dNTP pool, slow S-phase progression,

hypersensitivity to DNA damage, as well as DNA integrity checkpoint activation and

dependency. Additionally, these mutants have increased mutation rates, defective double-

strand break (DSB) repair by homologous recombination, and are completely unable to

undergo meiosis (Zolezzi et al., 2002; Liu et al., 2003, Liu et al., 2005; Holmberg et al.,

2005; Moss et al., 2010). Interestingly, loss of spd1 or overexpression of suc22 have been

shown to fully reverse the meiotic defects and checkpoint dependency of CRL4Cdt2

-

deficient cells (Holmberg et al., 2005; Liu et al., 2003; Yoshida et al., 2003). On the other

hand, the increased mutation rates and sensitivity to DNA damage are not completely

suppressed by spd1 deletion, indicating that other CRL4Cdt2

targets also contribute to the

observed phenotypes.

It has been commonly accepted, that all these effects are a consequence of lower

dNTP pool caused by Spd1-mediated RNR inhibition. However, recently it has been

demonstrated that Spd1 accumulation in cdc22-D57N RNR mutants generating higher

dNTP pools, still leads to checkpoint activation (Fleck et al., 2013). Moreover, survival of

cells which cannot degrade Spd1 is dependent on intact Rad3ATR

signaling even when they

are exogenously supplied with dNTPs (O. Nielsen, unpublished data). Together these

observations suggest that in addition to RNR, Spd1 has other targets of inhibition and its

constitutive levels interfere with yet unknown cellular pathway, leading to checkpoint

activation and dependency.


14

TORC2-Gad8 signaling in fission yeast

Target of rapamycin (TOR) is a highly conserved PIKK protein kinase that is a major

regulator of cell growth, proliferation and survival in response to environmental changes

and stress (Laplante and Sabatini, 2012). TOR takes part in the assembly of two

structurally and functionally distinct protein complexes, TORC1 and TORC2, which react

to different stimuli and target different set of substrates. TORC1 is inhibited by rapamycin,

while TORC2 is insensitive to the drug (Loewith et al., 2002).

Fission yeast cells contain two homologous genes encoding for TOR kinases – tor1

and tor2. Tor1 forms a complex with Ste20 and Sin1 to constitute TORC2, while Tor2

together with Mip1 form TORC1 (Matsuo et al., 2007; Hayashi et al., 2007). TORC2 is

not essential for growth in S. pombe, but is a major regulator of cellular response to a

variety of environmental stress factors (Cybulski and Hall, 2009).

TORC2 phosphorylates and activates the downstream kinase Gad8, which is the main

mediator of TORC2 function in fission yeast. Gad8 is a homologue of mammalian AKT1

kinase and belongs to the AGC subfamily of protein kinases (Matsuo et al., 2003; Ikeda et

al., 2008). TORC2 has pleiotropic functions and its activity is important for surviving

osmotic, oxidative and temperature changes, sexual development induced by starvation,

entry to mitosis, response to glucose availability, gene silencing, and telomere length

maintenance (Schonbrun et al., 2009; Schonbrun et al., 2013; Petersen and Nurse, 2007;

Cohen et al., 2014; Hatano et al., 2015). In addition, TORC2-Gad8 function is required for

surviving DNA damage, as deletion mutants of tor1 or gad8 are extremely sensitive to

HU-induced replication stress and DNA damage caused by exposure to MMS (Schonbrun

et al., 2009; Schonbrun et al., 2013). Hence, these mutants produce cellular phenotypes

highly similar to that of CRL4Cdt2

-deficient cells, suggesting that both TORC2-Gad8 and

CRL4Cdt2

complexes might function in the same pathway to preserve genome stability.


15

Results

Screen for spontaneous suppressors of Δcdt2 growth defects

To further our understanding of Spd1 function, a screen for spontaneous suppressors

of the physiological defects exhibited by CRL4Cdt2

-deficient S. pombe cells was

performed. This part of the work was executed by Adrian Ewald and presented in his

Master’s thesis (Ewald, 2013). Deletion mutants of cdt2 were grown for approximately

250 generations in two independent cultures and compared with equally treated wild type

cells for the emergence of suppressor mutations. Two single colonies were isolated from

each Δcdt2 replicate and analyzed by whole genome sequencing (Ewald, 2013).

Originally, it was anticipated that numerous mutations accumulated across the genome

would be detected, as cells with inactive CRL4Cdt2

pathway exhibit decreased dNTP levels

and elevated mutation rates (Moss et al., 2010; Holmberg et al., 2005). However, the

sequencing data revealed that each replicate had accumulated only two different mutations

when compared with wild type cells. Colonies from the first Δcdt2 replicate carried a

V40G substitution in the spd1 gene, and a guanine deletion in a TTG codon for leucine

725 in the uncharacterized SPCC4G3.12c gene (predicted to code for and E3 ubiquitin

ligase protein), leading to an early stop codon at amino acid position 745. Colonies from

the second Δcdt2 culture carried an S43L substitution in the spd1 gene, and a V335G

substitution in the uncharacterized SPCC16C4.02c gene, predicted to be an orthologue of

human neurochondrin (Ewald, 2013).

The observation, that Δcdt2 mutants did not accumulate numerous mutations in the

genome, suggests that the identified suppressors emerged early in the generations and

were sufficient to preserve the genome stability of the cells. These results suggest that V40

and S43 residues are important for the ability of Spd1 to confer genome instability in cells

with abrogated CRL4Cdt2

E3 ligase.

Furthermore, in a parallel study, we observed that Spd1 protein was phosphorylated in

vitro at S43 by Gad8 kinase, the fission yeast homologue of mammalian Akt1 (J. Petersen,

unpublished data). Interestingly, recent findings suggest that the TORC2-Gad8 complex is

important for genome integrity, since mutants with abrogated TORC2 signaling pathway

are extremely sensitive to replication stress and DNA damage (Schonbrun et al., 2013), a


16

phenotype exhibited also by CRL4Cdt2

defective cells. These findings indicate that the

cellular functions of Spd1 are possibly regulated by Gad8 kinase.

Collectively, our observations suggest that the V40 and S43 residues are important for

the function of Spd1 as inhibitor of cell cycle progression and that residue S43 is possibly

subject to post-translational modification by phosphorylation.

Amino acid residues V40 and S43 are important for the function of Spd1

To confirm the suppressive effect of the V40G and S43L mutations, each mutation

was introduced into Δddb1 background, and the phenotype of the double mutants was

further analyzed. Using a similar experimental approach, we also studied the functional

consequences of potential Spd1 phosphorylation by introducing a phosphomimetic and

non-phosphorylatable substitutions of residue S43.

The V40G, S43L and S43D mutations in spd1 suppress the checkpoint dependency of

Δddb1

The viability of cells with dysfunctional CRL4Cdt2

pathway depends on constitutive

activation of the Rad3ATR

checkpoint pathway. This phenotype is suppressed by

concomitant deletion of the spd1 gene (Holmberg et al., 2005; Liu et al., 2003),

suggesting that accumulation of Spd1 is a major cause for checkpoint activation. Mutants

carrying the rad3-ts allele have intact checkpoint signaling at 25 ºC, but are checkpoint-

deficient at 37 ºC. To test whether the V40G and S43L mutations were able to suppress

the requirement of Δddb1 cells for active Rad3ATR

pathway, each mutation was introduced

into Δddb1 rad3-ts genetic background. The viability of the triple mutants was analyzed at

the permissive and restrictive temperature. Indeed, the Δddb1 rad3-ts mutant was inviable

at 37 ºC. The spd1-S43L and spd1-V40G alleles suppressed the checkpoint requirement of

Δddb1 as effectively as spd1 deletion (Figure 3A and 3B). These observations suggest that

both V40G and S43L mutations inactivate the function of Spd1 and alleviate the

checkpoint dependency associated with Spd1 accumulation.

We next tested whether the phosphomimetic S43D substitution has an effect on the

checkpoint dependency of Δddb1 mutants. The spd1-S43D allele suppressed the synthetic

lethality conferred by loss of ddb1 and Rad3ATR

activity (Figure 3C). In contrast, Δddb1

rad3-ts cells carrying the non-phosphorylatable S43A mutation were completely inviable


17

at the restrictive temperature (Figure 3C). Thus, unlike S43A, the phosphomimetic S43D

mutation alleviates the checkpoint dependency conferred by excess Spd1, suggesting that

possible phosphorylation at S43 would serve to functionally inactivate Spd1.

Fig. 3. The V40G, S43L and S43D mutations of spd1 largely suppress the checkpoint dependency of Δddb1

cells. Tenfold serial dilutions of the indicated strains were spotted on YEA plates and incubated for 3 days at

the indicated temperatures. In accordance with previous data, deletion of spd1 rescues the viability of Δddb1

rad3-ts cells at the restrictive temperature (Holmberg et al., 2005). Similarly to the spd1 null allele, (A)

spd1-S43L and (B) spd1-V40G alleles suppress the checkpoint dependency of ddb1 deletion mutants. Two

independent clones of Δddb1 spd1-S43L rad3-ts and Δddb1 spd1-V40G rad3-ts were tested. (C) The

phosphomimetic S43D mutation alleviates the checkpoint dependency of Δddb1 mutants. The viability of

Δddb1 spd1-S43A cells is still dependent on active checkpoint signaling. Strains used: Eg 3156, Eg 3824, Eg

2634, Eg 3801, Eg 2736, Eg 2735, Eg 3795, Eg 3796, Eg 3512, Eg 3822, Eg 3798, Eg 3800, Eg 2738, Eg

3538, Eg 3464, Eg 3482, and Eg 3481.

DNA damage sensitivity of Δddb1 is partially suppressed by V40G, S43L and S43D

mutations in spd1

During S-phase and upon DNA damage Spd1 is targeted for degradation by CRL4Cdt2

ubiquitin ligase. Cells with inactive CRL4Cdt2

pathway are hypersensitive to DNA

damaging agents and replication stress. These phenotypes are partially suppressed by

concomitant deletion of the spd1 gene, suggesting that accumulation of Spd1 protein

contributes to the increased sensitivity to genotoxic stress (Liu et al., 2003; Holmberg et

al., 2005; Zolezzi et al., 2005; Vejrup-Hansen et al., 2014). Therefore, we investigated

whether the naturally selected V40G and S43L mutations could alleviate the sensitivity of


18

Δddb1 cells to DNA damage caused by the DNA alkylating agent methyl

methansulphonate (MMS). MMS sensitivity of Δddb1 was rescued to a modest extent in

both Δddb1 spd1-V40G and Δddb1 spd1-S43L double mutants (Figure 4A and 4B). We

also examined survival of cells treated with the radiomimetic DSB-inducing agent zeocin.

Accordingly, both spd1-V40G and spd1-S43L alleles suppressed the DNA damage

sensitivity of Δddb1 to almost the same extent as the spd1 null allele (Figure 4A and 4B).

We further analyzed the sensitivity to replication stress caused by the RNR inhibitor

hydroxyurea (HU). When introducing spd1-V40G or spd1-S34L mutations, the HU

sensitivity of Δddb1 cells was suppressed to the same extent as when deleting spd1, an

effect observed mostly at lower concentrations of HU (Figure 4A and 4B). These results

confirm that the V40G and S43L mutations somehow inactivate Spd1 and partially rescue

the sensitivity of CRL4Cdt2

-defective Δddb1 cells to genotoxic stress.

We next investigated whether the phosphomimetic spd1-S43D mutation could affect

the sensitivity of Δddb1 cells to DNA damage and replication stress. Survival rate of

MMS- and zeocin-treated Δddb1 spd1-S43D cells was increased when compared to the

Δddb1 single mutant (Figure 5A). Similarly, the Δddb1 spd1-S43D mutant was slightly

less sensitive to HU-induced replication stress than Δddb1 cells (Figure 5A). In contrast,

the Δddb1 spd1-S43A double mutant exhibited the same sensitivity to MMS, zeocin and

hydroxyurea as Δddb1 single mutant (Figure 5B). Hence, the phosphomimetic S43D

substitution partially rescued the sensitivity of Δddb1 to DNA damage and replication

stress, whereas the Δddb1 spd1-S43A mutant displayed the same sensitivity as Δddb1.

These findings suggest that phosphorylation of S43 might play a role in regulating Spd1

activity.


19

Fig. 4. The spd1-V40G and spd1-S43L mutations suppress the sensitivity of Δddb1 to DNA damaging

agents. Ten-fold serial dilutions of the indicated strains were spotted on YEA plates and plates containing

the indicated concentrations of MMS, zeocin and HU. Plates were incubated at 30 ºC for 3 days. (A) The

V40G and (B) S43L mutations of spd1 partially rescue the sensitivity of Δddb1 cells to DNA damaging

agents and HU-induced replication stress. Two independent clones of Δddb1 spd1-V40G and Δddb1 spd1-

S43L were tested. Strains used: Eg 3156, Eg 2447, Eg 1410, Eg 3824, Eg 2193, Eg 3794, Eg 3793, Eg 3512,

Eg 3797, and Eg 3799.


20

Fig. 5. Cell survival of Δddb1 spd1-S43D and Δddb1 spd1-S43A mutants in response to DNA damage and

replication stress. Ten-fold serial dilutions of the indicated strains were spotted on YEA plates and plates

containing the indicated concentrations of MMS, zeocin and HU. Plates were incubated at 30 ºC for 3 days.

(A) The phosphomimetic spd1-S43D mutation partially suppresses the sensitivity of Δddb1 mutants to DNA

damage and HU-induced replication stress. (B) Similarly to Δddb1, Δddb1 spd1-S43A mutant exhibits

impaired colony formation when treated with MMS, zeocin and HU. Two independent clones of Δddb1

spd1-S43D and Δddb1 spd1-S43A were tested. Strains used: Eg 3156, Eg 2447, Eg 1410, Eg 3575, Eg 2193,

Eg 3461, Eg 3478, Eg 3684, Eg 3467, and Eg 3468.


21

Meiotic defects of Δddb1 are suppressed by V40G, S43L and S43D mutations in spd1

In response to limited supply of nutrients, fission yeast cells shift from growth to

sexual development. First, diploid zygotes are formed by conjugation of two haploid cells,

followed by entry to meiosis and sporulation (Nielsen, 2004; Yamamoto, 2004; Davey,

1998). Deletion mutants of the CRL4Cdt2

ligase are completely unable to initiate

premeiotic S-phase and to undergo meiosis after conjugation. This phenotype is associated

with Spd1 accumulation as deleting spd1 alleviates the spore formation defect of Δddb1

(Holmberg et al., 2005). Therefore, we tested whether the naturally selected V40G and

S43L mutations suppress the meiotic defects exhibited by Δddb1 mutants. The percentage

of zygotes containing zero, one, two, three or four spores was scored. Consistent with

previous results, homothallic h90

Δddb1 cells could not form spores, and deleting spd1

rescued this defect to almost wild type level (>80 % four-spored asci). Introducing spd1-

V40G or spd1-S43L mutations into Δddb1 background recovered the meiotic efficiency of

the cells to the same extent as spd1 deletion (>80 % four-spored asci) (Figure 6, Table 1).

We also analyzed the effect of putative phosphorylation at S43 on the meiotic competence

of Δddb1 cells. The meiotic efficiency of the homothallic Δddb1 spd1-S43D mutant was

similar to the one observed in Δddb1 Δspd1 cells (>80 % asci with four spores) (Table 1,

Figure 6). By contrast, the majority of zygotes formed by Δddb1 spd1-S43A mutant

completely failed to develop spores (>90 % empty asci) (Table 1, Figure 6). These results

suggest that both V40G and S43L mutations alleviate the inhibition of premeiotic S-phase

caused by Spd1 accumulation. Additionally, this function of Spd1 might also be regulated

by phosphorylation of residue S43.

Fig. 6. Meiosis in Δddb1 is rescued by the V40G, S43L and S43D mutations in spd1. Δddb1 mutants are

completely unable to undergo meiosis. Similarly to Δspd1, spd1-V40G, spd1-S43L and spd1-S43D alleles

suppress the meiotic defects of homothallic h90

Δddb1 cells. Accumulation of Spd1-S43A inhibits meiosis

and Δddb1 spd1-S43A mutants form empty zygotes. h90

mutant strains of the indicated genotypes were

grown on medium inducing mating and meiotic differentiation. Photographs were taken after 3 days of

incubation at 25ºC. Strains used: Eg 337, Eg 3219, Eg 2358, Eg 3800, Eg 3877, Eg 3478, and Eg 3476.


22

Table 1. Meiosis in Δddb1 is restored by the V40G, S43L and S43D mutations in spd1

Genotype Spores: 0 1 2 3 4 n

wild type 2 0 2 1 95 169

Δddb1 93 7 0 0 0 152

Δddb1 Δspd1 5 2 4 2 87 256

Δddb1 spd1-V40G 5 4 8 2 81 236

Δddb1 spd1-S43L 6 1 2 3 87 220

Δddb1 spd1-S43D 6 0 6 3 85 214

Δddb1 spd1-S43A 91 9 0 0 0 111

Homothallic h90 strains of the indicated genotype were spotted on solid minimal medium to induce mating and

meiosis. Number of ascospores in zygotes was counted and the results are presented as percentage of the counted

zygotes (n). Strains used: Eg 282, Eg 3219, Eg 2358, Eg 3800, Eg 3877, Eg 3478, and Eg 3476.

Altogether, our findings indicate that Δddb1 spd1-V40G and Δddb1 spd1-S43L

mutants resemble the phenotype of Δddb1 Δspd1, suggesting that these mutations

inactivate the inhibitory effect of Spd1 accumulation on DNA synthesis. Moreover,

phosphorylation of S43 possibly modulates the function of Spd1.

The phosphomimetic S43D mutation does not affect the stability of Spd1 protein

Spd1 is targeted for degradation by the CRL4Cdt2

E3 ligase during DNA synthesis and

following hydroxyurea treatment (Liu et al., 2005; Holmberg et al., 2005). To test whether

the stability of Spd1 is affected by the above analyzed V40 and S43 substitutions, levels of

mutant Spd1 protein were monitored by Western blot before and after HU addition. Due to

sudden loss of antibody activity, only the degradation pattern of Spd1-S43D protein was

followed successfully. Similar to the wild type protein, Spd1-S43D was degraded in

response to HU-induced replication stress. This response was completely abolished when

the function of the CRL4Cdt2

ubiquitin ligase was abrogated by deletion of ddb1 (Figure 7).

Hence, the phosphomimetic mutation of Spd1 does not change its stability, and Spd1-

S43D protein is degraded in the same pattern as wild type Spd1. These results suggest that

the S43D mutation suppresses the defects associated with loss of ddb1 by somehow

inactivating the function of Spd1, rather than decreasing its stability.


23

Fig. 7. The phosphomimetic S43D mutation does not affect the stability of Spd1 protein. Exponentially

growing cells of the indicated strains were harvested before and after treatment with 20 mM HU for 2 and 4

hours. TCA protein extracts were prepared and Spd1 levels were monitored by Western blot using

polyclonal antibody raised against recombinant Spd1. Wild type Spd1 and Spd1-S43D proteins were

degraded upon HU-induced replication stress. Spd1-S43D was stabilized in CRL4Cdt2

-defective Δddb1 cells.

Tubulin was used as a loading control. The size of the bands (kDa) is estimated according to the molecular

mass marker used in the experiment. Film was developed with the help of L. Langholm. Strains used: Eg

3156, Eg 3575, and Eg 3461.

Suc22R2

nuclear import function of Spd1 is not significantly affected by the V40G,

S43L and S43D mutations

Spd1 inhibits RNR activity outside S-phase by sequestering Suc22R2

in the nucleus,

thus preventing its interaction with Cdc22R1

, which is mainly cytosolic (Liu et al., 2003).

Most asynchronous fission yeast cells are in G2 and in wild type cells Suc22R2

accumulates predominantly in the nucleus. During S-phase or following DNA damage,

Suc22R2

is re-localized to the cytoplasm to promote formation of active RNR complexes

and dNTP synthesis. Deletion of spd1 or hydroxyurea-induced degradation of Spd1, result

in pan-cellular distribution of Suc22R2

(Liu et al., 2003; Nestoras et al., 2010). Amino acid

residues V40 and S43 reside in the in the Hug domain of Spd1, which is required for

interaction with Suc22R2

(Lee et al., 2008; Nestoras et al., 2010). Therefore, we next

tested whether the V40G and S43L mutations suppress the defective phenotypes of

CRL4Cdt2

-deficienct cells by interfering with the Suc22R2

nuclear import function of Spd1.

We constructed strains expressing spd1-V40G and spd1-S43L in GFP-suc22 background,

and the localization of Suc22R2

was observed by direct fluorescence in cells before or after

treatment with HU. Similarly to wild type, in spd1-S43L mutant Suc22R2

was localized

predominantly in the nucleus in non-S-phase cells, whereas it became largely cytosolic

upon HU treatment (Figure 8). Interestingly, the spd1-V40G mutant displayed a slight

defect in Suc22R2

nuclear accumulation, since we observed increased cytoplasmic

fluorescence compared to wild type cells (Figure 8). Although this small defect, the V40G


24

and S43L substitutions do not appear to have a significant effect on Suc22R2

nuclear

import function of Spd1. Hence, these mutations suppress the defects of CRL4Cdt2

-inactive

cells by affecting another function of Spd1, different from inhibiting RNR activity. This

notion is in accordance with previous reports that Spd1 accumulation in CRL4Cdt2

mutants

causes genome instability independently of RNR inhibition (Fleck et al., 2013).

Using the same approach, we further analyzed whether the phosphomimetic S43

mutation affects the nuclear accumulation of Suc22R2

. The profile of Suc22R2

subcellular

localization was not changed in mutants bearing the S43D substitution when compared to

wild type cells (Figure 8). Interestingly, whereas the spd1-S43A mutant was competent for

Suc22R2

nuclear import, it displayed a considerable defect in Suc22R2

cytoplasmic release

in response to HU treatment (Figure 8). These results suggest that phosphorylation of

Spd1 is possibly required for nuclear release of the small RNR subunit, since the inability

to phosphorylate Spd1 appears to prevent Suc22R2

delocalization to the cytoplasm.

Fig. 8. Subcellular localization of Suc22R2

in mutants bearing the V40G, S43L, S43D and S43A mutations

in spd1. Exponentially growing cells with the indicated genotypes and expressing GFP-Suc22R2

were treated

with 20 mM HU to arrest cells in S-phase and to promote Spd1 degradation. GFP-Suc22R2

was visualized by

direct fluorescence before and after treatment with HU. Consistent with previous data (Liu et al., 2003), in

wild type cells Suc22R2

localizes in the nucleus and upon HU treatment, GFP-Suc22R2

re-localizes to the

cytoplasm. In the absence of Spd1, GFP-Suc22R2

is constitutively spread all over the cell throughout the cell

cycle (Liu et al., 2003). Similarly to the wild type cells, in cells expressing spd1-S43L or spd1-S43D,

Suc22R2

is localized in the nucleus and distributes to the cytoplasm following treatment with HU. The spd1-

V40G mutant is slightly defective in Suc22R2

nuclear accumulation. The spd1-S43A mutant considerably

retains Suc22R2

in the nucleus after treatment with HU. Strains used: OL 1696, Eg 3340, Eg 3949, Eg 3963,

Eg 3349, and Eg 3355.


25

Spd1 amino acid residues V40 and S43 are important for interaction with PCNA

CRL4Cdt2

-dependent degradation of Spd1 requires interaction with DNA-loaded

PCNA. Spd1-PCNA interaction has been observed in vivo only when Spd1 is stabilized by

deletion of cdt2 (Salguero et al., 2012). Amino acid residues V40 and S43 are in close

proximity to the PIP degron of Spd1 which is important for interaction with PCNA. To

address whether the V40G and S43L mutations affect Spd1 binding to PCNA, we used

bimolecular fluorescence complementation (BiFC) (Akman and MacNeill, 2009; Hu and

Kerppola, 2003). We constructed strains in which PCNA and Spd1 (wild type or mutant)

were tagged with the N- and C-terminal domains of Venus-YFP respectively, and both

proteins were expressed from their endogenous promoters (Salguero et al., 2012).

Additionally, in all these strains transcription of cdt2 was under the control of the thiamine

repressible nmt41 promoter, which allowed us to shut off cdt2 expression by supplying

thiamine to the medium. In all strains no nuclear fluorescence was observed under cdt2-

inducible conditions (Figure 9, upper panels). When the turnover of wild type Spd1 was

blocked by depletion of Cdt2, YFP fluorescence was detected as punctate nuclear foci

(Figure 9). In spd1-S43L mutants no fluorescence was seen even when Cdt2 expression

was shut off. Interestingly, in spd1-V40G mutants YFP fluorescence was detected under

cdt2-repressible conditions, but the nuclear BiFC signal was diffuse and spread all over

the nucleus (Figure 9). Previously, similar punctate and diffuse nuclear GFP-PCNA signal

has been described, representing the chromatin-loaded (part of the replication factories)

and unloaded fraction of PCNA respectively (Meister et al., 2007). It is very probable that

our observations also correspond to the DNA-bound and free fraction of PCNA. Hence,

while the V40G and S43L mutations did not have a significant effect on Suc22R2

nuclear

accumulation, they did affect the interaction between Spd1 and PCNA. The S43L

substitution perturbs Spd1-PCNA interaction, whereas the Spd1-V40G protein appears to

retain binding only to the unloaded fraction of PCNA. In this study we demonstrated that

either of these mutations suppressed the defective phenotypes of CRL4Cdt2

-deficient cells,

but they did not have a dramatic effect on Suc22R2

nuclear sequestration. Thus, it is likely

that this suppression is due to the perturbed Spd1-PCNADNA

interaction, suggesting that

Spd1 possibly interferes with DNA synthesis by inhibiting the function of PCNA.


26

In this respect, Spd1 might share functional similarities with mammalian p21, which

is an intrinsically disordered protein known to bind strongly and inhibit PCNA by

preventing access for other interacting partners required during DNA synthesis (Waga et

al., 1994; Cazzalini et al., 2003). Furthermore, p21-PCNA interaction is released by Akt1-

mediated phosphorylation of p21 at amino acid residues residing within its PIP degron

(Scott et al., 2000; Rössig et al., 2001). Therefore, we next tested whether the

phosphomimetic S43D substitution would also prevent Spd1-PCNA interaction. Indeed,

no YFP signal was seen in the presence of the spd1-S43D mutation when cells were grown

under conditions repressing Cdt2 expression (Figure 9). Conversely, YFP fluorescence

like punctate nuclear foci was detected upon Cdt2 depletion in cells carrying the non-

phosphorylatable S34A mutation (Figure 9). These results suggest that phosphorylation of

Spd1 might be required to release the binding of Spd1 to PCNA, since the interaction

between both proteins is detected in the presence of the non-phosphorylatable S43A

mutation, but not in cells carrying the phosphomimetic S43D mutation.

Fig. 9. Spd1 residues V40 and S43 are important for Spd1-PCNA interaction. Interaction between Spd1 and

PCNA was analyzed by bimolecular fluorescence complementation (BiFC). Strains expressing VC173-pcn1

and wild type or mutant (V40G, S43L, S43D or S43A) spd1 –VC155 in nmt41-cdt2 background were used.

Cells were grown in the absence or presence of thiamine to induce or repress cdt2 expression respectively.

No nuclear fluorescence was observed in all strains when cells were grown under cdt2-inducible conditions

(upper panels). Upon depletion of Cdt2, nuclear YFP signal was detected as defined foci in cells expressing

wild type Spd1 or Spd1-S43A. No BiFC signal was observed in spd1-S43L and spd1-S43D mutants under

cdt2-repressible conditions. Diffuse nuclear YFP signal was detected in spd1-V40G cells when cdt2

expression was shut off. Strains used: Eg 3653, Eg 3827, Eg 3991, Eg 3828,and Eg 3986.


27

Deletion of spd1 does not suppress the sensitivity of gad8 mutants to replication stress

and DNA damage

As mentioned earlier, the TORC2 complex is required for genome integrity in fission

yeast and disruption of the TORC2-Gad8 pathway produces phenotype which highly

resembles the phenotype of CRL4Cdt2

deficient cells. Similarly to Δddb1, deletion of tor1

or gad8 adversely affects the cellular sensitivity to HU-induced replication stress and

DNA damage (Schonbrun et al., 2013). In addition, our studies demonstrate that Spd1 is

phosphorylated in vitro by Gad8 (the fission yeast homologue of mammalian Akt1 kinase)

at S43 (J. Petersen, unpublished data). Our attempts to detect Spd1 phosphorylation in

vivo were not successful, due to the low specificity of the Spd1-S43-phosphospecific

antibody when used on whole cellular extracts (L. Langholm, data not shown). Therefore,

we tested whether the defective phenotypes of TORC2-Gad8-deficient cells are due to the

inability to phosphorylate Spd1. We deleted the spd1 gene in Gad8 kinase-dead gad8-

K259R mutants (Matsuo et al., 2003) and analyzed the viability of the double mutants in

response to replication stress and DNA damage. Since Gad8-deficient cells are genetically

unstable and prone to accumulate suppressor mutations, six individual gad8-K259R Δspd1

colonies were tested in this assay. Consistent with previous data (Schonbrun et al., 2013),

gad8-K259R cells were extremely sensitive to treatment with HU and MMS (Figure 10).

These phenotypes were not suppressed by concomitant deletion of spd1 (Figure 10).

Hence, the drug sensitivity of gad8-K259R cells is not due to the inability to

phosphorylate Spd1, suggesting that Spd1 is not the only target of Gad8. Since possible

regulation of Spd1 by phosphorylation still needs to be detected in vivo, we cannot rule

out the possibility that Spd1 is phosphorylated by another cellular kinase having

overlapping functions with Gad8 regarding particular substrates. Based on these results we

cannot conclude whether Spd1 is a target of the TORC1-Gad8 pathway.


28

Fig. 10. Deletion of spd1 does not suppress the sensitivity of TORC2-Gad8 defective cells to replication

stress and DNA damage. Strains were grown to logarithmic stage at 30ºC. Sequential ten-fold serial dilution

of cells of each strain was spotted on YEA plates containing the indicated concentration of HU and MMS.

Plates were incubated at 30 ºC for three days. Strains used: Eg 3156, Eg 3597, three colonies - streak from

Eg 3892, three colonies - streak from Eg 3893.

Overexpression of suc22 affects the interaction between Spd1 and PCNA

The defective phenotypes of cells with compromised CRL4Cdt2

pathway are largely

suppressed by deletion of spd1, or alternatively, by overexpression of suc22 (Liu et al.,

2003; Yoshida et al., 2003; Holmberg et al., 2005). Furthermore, elevated levels of

Cdc22R1

do not rescue the defects of Δcdt2 mutants (Yoshida et al., 2003). A commonly

accepted interpretation of these observations has been that Spd1 accumulation restrains

RNR activity by sequestering the small RNR subunit in the nucleus, which eventually

leads to checkpoint activation due to insufficient dNTPs levels during DNA synthesis.

Thus, higher Suc22R2

levels serve to counterbalance the lower cytoplasmic abundance of

Suc22R2

in CRL4Cdt2

mutants, suppressing the phenotypes conferred by excess Spd1.

However, in this study we showed that the suppressor V40G and S43L mutations mainly

affect the binding of Spd1 to PCNA. Therefore, using the above mentioned BiFC system,

we analyzed whether overexpression of suc22 would also have an effect on Spd1-PCNA

interaction. As expected, we observed punctate nuclear YFP foci upon Cdt2 depletion in

cells expressing wild type levels of Suc22R2

(Figure 11). Interestingly, when suc22

expression was driven by the constitutively active ADH promoter, we detected diffuse

nuclear BiFC signal (Figure 11), similar to the one observed in spd1-V40G mutants. These

results confirm, that excess levels of Suc22R2

somehow titrate out Spd1 from PCNADNA

and support the hypothesis that Spd1 functions to inhibit PCNA.


29

Fig. 11. Overexpression of suc22 affects the interaction between Spd1 and PCNA. Interaction between Spd1

and PCNA was analyzed by bimolecular fluorescence complementation (BiFC) as described in Figure 9.

The constitutively active ADH promoter was used to overexpress suc22. No nuclear fluorescence was

detected when Cdt2 was expressed (upper panels). Define punctate nuclear YFP signal was detected in

strains expressing wild type levels of Suc22 upon depletion of Cdt2. Diffuse nuclear BiFC signal was

observed in strains with elevated levels of Suc22. Strains used: Eg 3945 and Eg 3947.

Discussion

The fission yeast Spd1 protein has a significant impact on genome integrity, but its

biological function is not fully understood. In this study we provide additional insights

into the function and regulation of Spd1-mediated interactions. We show that the naturally

selected V40G and S43L mutations, as well as the phosphomimetic S43D substitution,

suppress the checkpoint dependency, the DNA damage sensitivity and the meiotic defects

of Δddb1 cells as efficiently as spd1 deletion. Conversely, mutants carrying the non-

phosphorylatable S43A substitution in Δddb1 background are completely dependent on

intact DNA integrity checkpoint signaling, exhibit high sensitivity to DNA damage and

are unable to undergo meiosis. These results suggest that the V40 and S43 residues of

Spd1 are critical for its function as an inhibitor of DNA synthesis during S-phase and

DNA repair.

It is not likely that the analyzed mutations of these residues affect the stability of

Spd1. As we show here, the degradation pattern of Spd1-S43D protein is similar to the

wild type protein, whereas in Δddb1 spd1-S43D mutants Spd1-S43D levels remain


30

persistent even after treatment with HU. Additionally, previous in vitro experiments with

mutant Spd1-S43A protein do not suggest any evidence of increased stability of the

protein (Nestoras et al., 2010). We conclude that the analyzed substitutions of residues

V40 and S43, do not change the stability of Spd1, but rather interfere with interactions

between Spd1 and some specific partners.

Interestingly, amino acid residues V40 and S43 are adjacent to the PIP degron motif

of Spd1, which resides in the Hug domain of the protein (Figure 2B). While the PIP

degron mediates binding of Spd1 to the DNA polymerase processivity factor (Salguero et

al., 2012), the Hug domain is supposed to be required for interaction with the small RNR

subunit (Lee et al., 2008; Nestoras et al., 2010). Therefore, we assume that mutations of

these residues would affect the interaction of Spd1 with both Suc22R2

and PCNA. We

demonstrate that the S43L and S43D mutations retain the nuclear import function of Spd1

and do not affect the pattern of Suc22R2

subcellular localization following HU treatment.

However, these mutations appear to prevent the interaction between Spd1 and PCNA,

since we do not detect nuclear BiFC fluorescence even upon stabilization of Spd1 by Cdt2

depletion. Interestingly, the V40G substitution appears also to weaken the interaction

between Spd1 and Suc22R2

, which is suggested by the small but notable defect on Suc22R2

nuclear accumulation. Additionally, this mutation also affects the mode of Spd1-PCNA

interaction, giving rise to diffuse nuclear YFP signal, most probably representing binding

of Spd1 to the DNA-unloaded fraction of PCNA. In contrast, the S43A substitution

appears to confer strong binding to Suc22R2

, since mutants bearing this mutation display

limited cytoplasmic delocalization of the small RNR subunit upon HU treatment.

Moreover, the non-phosphorylatable form of Spd1 retains the ability to bind PCNA like

wild-type Spd1, an interaction observed like punctate nuclear YFP foci, probably

representing the chromatin-loaded portion of active PCNA, participating in the formation

of replication factories (Meister et al., 2007) . These findings indicate that the V40 and

S43 amino acid residues of Spd1 are important for its interaction with both, Suc22R2

and

PCNA.

The substitutions V40G, S43L and S43D, which we showed to alleviate the defective

phenotypes of Δddb1 cells, affect most significantly the Spd1-PCNA interaction. These

substitutions either totally prevent the interaction between Spd1 and nuclear PCNA, or

allow binding only between Spd1 and DNA-unloaded PCNA. Conversely, the S43A


31

mutation, which retains the harmful effect of Spd1 accumulation and its ability to bind

PCNADNA

, additionally retains the small RNR subunit in the cytoplasm. Based on these

findings, we suggest that stabilization of the Spd1-PCNADNA

interaction inhibits the

activity of the DNA polymerase processivity factor and presumably leads to checkpoint

activation.

This assumption is further supported by the observation that overexpression of

Suc22R2

, which is known to suppress the phenotypes associated with inactive CRL4Cdt2

pathway (Liu et al., 2003; Yoshida et al., 2003; Holmberg et al., 2005) also affects Spd1-

PCNA interaction, resulting in diffuse nuclear BiFC foci. Thus, elevated levels of Suc22R2

appear to somehow prevent the interaction between Spd1 and PCNADNA

. These data

suggest that when Suc22R2

is overexpressed, it probably competes out the binding of Spd1

to PCNADNA

, thus alleviating the checkpoint dependency caused by Spd1 accumulation.

Based on our current results, we propose that the nuclear pool of Spd1 forms separate

complexes with Suc22R2

and PCNA, as well as tertiary PCNA-Spd1-Suc22R2

complexes.

We assume that the checkpoint activation and the defective phenotypes caused by

accumulation of Spd1 are due to its inhibitory interaction with the chromatin-bound

PCNA represented by PCNADNA

-Spd1 or PCNADNA

-Spd1-Suc22R2

complexes, rather than

the nuclear sequestration of Suc22R2

. It is possible that Suc22R2

is required in the nucleus

to increase the nuclear dNTP supply, as well as to assist the formation of active PCNA

complexes. A nuclear function of Suc22R2

is not surprising, as previous reports also

suggest the need for RNR activity in the nucleus where it might be as well, physically

linked to sites of DSB repair (Niida et al., 2010; Moss et al., 2010). In this context, Spd1

might serve to recruit the small RNR subunit in the nucleus and to regulate the formation

of active Suc22R2

-PCNA and Suc22R2

-Cdc22R1

complexes for DNA synthesis according

to the physiological requirements of the cell. Presumably, such mechanism would provide

the cell with rapid and flexible response to DNA damage outside S-phase.

The small but notable defect of spd1-V40G mutants in Suc22R2

nuclear accumulation

might be due to weaker Spd1-Suc22R2

binding, resulting in easier dissociation of Spd1-

V40G-Suc22R2

complexes. This assumption is supported by structural analysis showing

that Spd1-V40G binds PCNA with the same affinity as wild-type Spd1, whereas it

exhibits lower affinity binding towards Suc22R2

(Birthe Kragelund, personal


32

communication). Thus, free nuclear Suc22R2

probably releases the inhibition of PCNA by

titrating out Spd1-V40G from PCNADNA

.

Although functional homologues of Spd1 have not been identified in mammalian

cells, due to limited sequence conservation, our observations suggest that Spd1 might

share some functional similarities with p21. AKT1-mediated phosphorylation of p21 is

required to release the inhibitory binding of p21 to PCNA, and Spd1 might be regulated in

a similar fashion. However, at the current stage of our study we cannot conclude whether

this regulatory mechanism also exists in fission yeast. Our attempts to detect Spd1

phosphorylation in vivo were not successful, and deletion of spd1 does not alleviate the

DNA damage sensitivity of Gad8 kinase-dead mutants, suggesting that Spd1 is not the

only target of this kinase. Hence, further work is required to establish whether this post-

translational modification occurs in vivo.

The work on this study is still in progress and the fidelity of the above mentioned

hypothesis awaits further investigation. We do not rule out the possibility that other

interacting partners might be involved in it. Most importantly, the interaction between

PCNA and Suc22R2

in live cells needs to be validated. Additionally, the binding affinities

and the stoichiometry of the different interactions should also be determined. Considering

the low specificity of the S43-phosphospecific antibody, enrichment of phosphorylated

cellular proteins followed by mass spectrometry analysis would shed light on the

phosphorylation status of Spd1.


33

Chapter 2

Screen for Spontaneous Suppressors of the

Fission Yeast Temperature Sensitive cdc48-353

Mutant

Chapter objectives:

Describes a strategy for the

identification of pseudo revertants of

the temperature sensitive cdc48-353

fission yeast mutant using whole

genome sequencing approach.


34


35

Introduction

Cdc48/p97 is an essential ring-shaped homohexameric chaperone-like complex

involved in numerous cellular processes, including protein degradation, cell cycle control,

DNA repair, and vesicle fusion. Although the activity of Cdc48/p97 has been extensively

investigated, it is still not clear why its function is essential for the cells. The aim of this

project was to provide further insight into the interaction network of Cdc48/p97 in fission

yeast. In this study we performed a screen for pseudo revertants of the cdc48-353

temperature-sensitive (ts) fission yeast mutant. Using whole genome sequencing approach

we identified 28 independent spontaneous suppressors of the ts growth defects exhibited

by the cdc48-353 strain, which had also acquired a cold-sensitive (cs) phenotype.

Structural analysis revealed that whereas the original G338D ts lesion was located near the

central pore of the hexameric ring, most of the suppressor mutations were positioned at the

subunit-subunit boundaries. Our observations suggest that the structure of the Cdc48-353

hexamer is destabilized at the restrictive temperature and re-stabilized in the suppressor

mutants. In addition, we isolated a suppressor which had not acquired a cs phenotype.

Whole genome sequencing analysis revealed an additional frame shift mutation leading to

a stop codon in the carboxy-terminal half of Ufd1, which is a known co-factor of Cdc48.

The broad activity of Cdc48/p97 will be reviewed in the following section. First, the

structural organization of the Cdc48/p97 hexamer will be introduced, followed by a

review of its assembly with different interacting partners. Special emphasis is put on

Cdc48Ufd1-Npl4

function in the cell cycle control and maintenance of genome integrity.

Structure and function of Cdc48/p97

Cdc48 is an essential highly conserved member of the AAA-ATPases family also

known as p97 or VCP (vasolin-containing protein) in higher eukaryotes (Stolz et al.,

2011). Cdc48 is a chaperone-like complex composed of six monomers assembled in two

stacked homohexameric rings surrounding a central cavity (Stolz et al., 2011). Each

monomer contains an N-terminal domain, two ATPase domains (D1 and D2) and a

disordered C-terminal region. The N-terminal domain is required for interaction with most

of the Cdc48 co-factors (Buchberger et al., 2015). The D1 and D2 modules sustain the

architecture of the hexameric ring and are responsible for binding and hydrolysis of ATP

Screen for Spontaneous Suppressors of the Fission Yeast Temperature Sensitive cdc48-35 Mutant

36

(Stolz et al., 2011). The energy derived from ATP hydrolysis induces extensive

conformational rearrangements to the whole ring complex (DeLaBarre and Brunger, 2006;

Briggs et al., 2008). These rearrangements are necessary for the mechanistic function of

Cdc48 to segregate specific proteins from intracellular structures (Beuron et al., 2006).

Cdc48 is one of the most abundant proteins in the cell (Peters et al., 1990) and serves

as a central factor in the execution of cellular processes mediated my ubiquitin or

ubiquitin-like proteins. The diverse molecular activities of Cdc48 are controlled by a large

number of co-factors which provide specificity towards particular cellular pathway (Franz

et al., 2016b; Xia et al., 2016). Most cofactors bind Cdc48 through conserved modules

and can be functionally divided into two groups – substrate recruiting factors and substrate

processing factors (Buchberger et al., 2015). Substrate recruiting cofactors direct Cdc48 to

specific cellular locations by recognizing ubiquitylated or SUMOylated protein targets.

Processing cofactors can modify the length and topology of the ubiquitin and SUMO

conjugates thus influencing the fate of Cdc48 substrates (Buchberger et al., 2015).

Importantly, biochemical and structural studies have demonstrated that some co-factors

interact with Cdc48 in a competitive manner as they bind partially overlapping regions of

Cdc48 (Hӓnzelmann and Schindelin, 2011), suggesting that different Cdc48 complexes

may exist in the cell, each executing distinct activities. Cdc48/p97 function is essential for

viability and mutations or changes in Cdc48/p97 protein expression in humans are

associated with neurological disorders, premature aging and cancer (Nalbandian et al.,

2011; Johnson et al., 2010; Franz et al., 2014; Fessart et al., 2013).

One of the best characterized assemblies of Cdc48 is in a complex with the adapter

protein p47. p47 interacts with Cdc48 through a UBX domain (Dreveny et al., 2004) or

SHP box motif (Bruderer et al., 2004) and directs Cdc48 towards monoubiquitylated

target proteins. Together with other partners, p97p47

complex has been implicated in

playing an essential role in autophagy (Krick et al., 2010) and reassembly of the Golgi

apparatus after mitosis (Kondo et al., 1997). In addition, p97 and the cofactor UBXD7

have been reported to participate in the ubiquitin-mediated turnover of hypoxia-inducible

factor 1α (HIF1α), where UBXD7 directly links p97 to the ubiquitin ligase CUL2/VHL

and its substrate HIF1α (Alexandru et al., 2008).

The heterodimer Ufd1/Npl4 serves as major adaptor of Cdc48 and is broadly involved

in assisting the functions of the hexameric chaperone. Npl4 is an essential protein (Kim et


37

al., 2010), which interacts with Cdc48 via a UBXL domain (Buchberger et al., 2015).

Ufd1 is also essential for cell viability (Kim et al., 2010) and its N-terminal part contains

mono- and polyubiquitin binding sites (Park et al., 2005). The C-terminal part of Ufd1

comprises a SHP box required for interaction with Cdc48 and a SUMO interacting motif

(SIM) (Buchberger et al., 2015). The best characterized function of Cdc48Ufd1-Npl4

complex is in the endoplasmic reticulum-associated degradation (ERAD) of misfolded

proteins. Cdc48Ufd1-Npl4

is recruited to the ER membrane through interaction with the

membrane-bound protein Ubx2 (Schuberth and Buchberger, 2005). Thus membrane-

associated Cdc48 binds ubiquitylated substrates emerging from the retrotranslocation

channel. Energy derived from ATP hydrolysis is utilized for the segregation of misfolded

proteins from the ER membrane, which are then targeted for proteasomal degradation (Ye

et al., 2001; Braun et al., 2002). Ufd1/Npl4 adaptor is also involved in the degradation of

aberrant nascent polypeptides bound to the ribosome. Cdc48, Npl4, Ufd1 and other

interacting partners are highly enriched on the 60S ribosomal subunit where they are

suggested to assist the release of ubiquitylated polypeptides thus targeting them for

proteasomal destruction (Defenouillere et al., 2013; Verma et al., 2013). Besides

functioning in proteasome-dependent processes, p97Ufd1-Npl4

complex also plays a critical

role in regulation of mitochondrial homeostasis. p97, Ufd1 and Npl4 localize on the

surface of damaged mitochondria and silencing of either Npl4 or Ufd1 is demonstrated to

cause impaired clearance of damaged mitochondria in flies (Kimura et al., 2013).

Additionally, p97Ufd1-Npl4

complex participates in the mobilization of dormant transcription

factors where it promotes the segregation of certain ER membrane anchored proteins via a

polyubiquitin signal. The released transcription factors are subsequently transported to the

nucleus to execute their function (Rape et al., 2001; Shcherbik and Haines, 2007).

The essential cellular activities of Cdc48/p97 also include regulation of cell cycle

progression and maintenance of genome integrity, but the precise mechanism for these

processes is still elusive. Cdc48 was originally identified as a factor controlling the cell

division cycle in yeast (Moir et al., 1982), but its exact role in cell cycle regulation is still

unclear. Cdc48 plays a critical role in mitosis, since conditional cdc48 mutants are

defective in G2/M transition (Moir et al., 1982). In addition, Cdc48 is required for passing

the Start point in budding yeast after mitosis is completed, as it mediates the degradation

of the G1-CDK inhibitor Far1 (Fu et al., 2003). Moreover, fission yeast cells bearing a


38

temperature sensitive cdc48 allele lose viability in anaphase at the restrictive temperature.

Presumably, Cdc48 promotes proper segregation of sister chromatids by stabilizing

Cut1/separase during transition from metaphase to anaphase (Ikai and Yanagida, 2006).

Cdc48/p97 is also engaged in the regulation of chromatin abundance of Aurora B kinase

in mitosis and meiosis (Ramadan et al., 2007; Sasagawa et al., 2012). Ubiquitylated

Aurora B kinase is extracted from chromatin by Cdc48/p97 associated with Ufd1-Npl4,

which results in chromosome decondensation (Ramadan et al., 2007). However, it is still

not clear whether Aurora B kinase is subsequently degraded by the proteasome as its total

protein levels are not affected by depletion of p97 (Sasagawa et al., 2012).

The p97Ufd1-Npl4

complex is essential for DNA replication. RNAi mediated

downregulation of p97, Ufd1 or Npl4 results in delay of cell cycle progression, checkpoint

activation and formation of DNA repair foci (Mouysset et al., 2008). During replication

and after DNA damage, Cdc48Ufd1-Npl4

in concert with the substrate recognition module

UBXN3/FAF1 prevents re-replication and ensures proper fork progression by extracting

the replication licensing factor Cdt1 from chromatin (Raman et al., 2011; Franz et al.,

2016a). Moreover, it has been demonstrated that Cdc48/p97 activity is also required for

the release of the CMG complex, the eukaryotic replicative helicase, at the final stages of

DNA replication (Maric et al., 2014). However, the precise role of Cdc48 during DNA

synthesis is not clear, as different co-factors and different evolutionary mechanisms might

exist in different kingdoms to control timely release of Cdt1 and CMG from the chromatin

(Franz et al., 2016b).

Bulky lesions induced by UV irradiation are a serious threat for genome integrity as

they interfere with transcription by stalling RNA polymerase II. UV-induced DNA

damage is detected by XPC and DDB1-DDB2 complex followed by initiation of repair

through nucleotide excision repair (NER) pathway. Cdc48/p97 and the cofactors Ufd1-

Npl4 ensure proper execution of the repair process by ubiquitin-dependent extraction of

XPC, DDB2 and Rbp1 (the largest subunit of RNA polymerase holoenzyme) from the

sites of damage (Puumalainen et al., 2014; Verma et al., 2011). The broad activity of

Cdc48/p97 also extends to repair of DNA double strand breaks (DSB). Recent

observations indicate that Cdc48 facilitates DSB repair by removal of ubiquitylated

proteins from DNA break sites and, presumably, recruiting downstream repair factors,

even though the detailed mechanism of this activity is still unclear (Meerang et al., 2011).


39

In addition to ubiquitin-conjugated proteins, CDC48/p97 is also involved in extraction of

SUMOylated targets from chromatin during DSB repair through homologous

recombination. In this process, the co-factor Ufd1 plays a central role. Ufd1 binds SUMO

conjugates directly via a SUMO interaction motif (SIM) thus recruiting Cdc48 at the sites

of damage. Furthermore, Ufd1 interacts with SUMO-targeted ubiquitin ligases to promote

ubiquitylation and, presumably, degradation of specific substrates (Nie et al., 2012).

Results and Discussion

To gain deeper insight into the interaction network of Cdc48, we performed a screen

for spontaneous cold sensitive (cs) suppressors of the temperature sensitive (ts) cdc48-353

mutant in fission yeast (Yuasa, et al., 2004; Ikai and Yanagida, 2006), which carries a

G338D substitution in the conserved D1 domain. Altogether we isolated 29 independent

pseudo revertants that had lost the temperature sensitivity of the original ts mutant. 28 of

the suppressors (named cdc48-353sup

-1-28) had also acquired a cs phenotype, whereas

mutant cdc48-353sup

-29 appeared not to be cs upon retesting. Using a whole genome

sequencing strategy followed by subsequent genetic analysis, we found that the cs cdc48-

353sup

-1-28 suppressors were all second site intragenic cdc48 mutants. Sequencing results

revealed that suppressor cdc48-353sup

-29 carries a frame shift mutation in the ufd1 gene

resulting in an early stop codon, while the cdc48-353 allele was not changed (Marinova et

al., 2015).

Mapping the position of the intra-cdc48 ts suppressing variants on the domain

organization of Cdc48 revealed that most of the suppressor mutations were located at the

C-terminal part of Cdc48 containing the D1 and D2 ATPase domains. The N-terminal

domain was not directly affected in any of the identified mutations. Whereas the original

G338D ts lesion is positioned in the D1 domain facing the central pore of the hexameric

ring, most of the suppressor mutations are located at the subunit-subunit interfaces and in

loops between the domains. This suggests that the original G338D substitution probably

destabilizes the hexameric ring structure of Cdc48 at the restrictive temperature and that

the intragenic suppressor mutations compensate this by enhancing the contacts at subunit-

subunit interfaces. Therefore under cold conditions the cs cdc48 suppressors might exhibit

decreased flexibility of the Cdc48 hexamer resulting in compromised chaperone function

(Marinova et al., 2015).


40

The ufd1 gene is essential in fission yeast (Kim et al., 2010) and it is only known to

function as a cofactor of Cdc48. The frame shift mutation in ufd1 identified in suppressor

cdc48-353sup

-29 leads to a deletion of approximately 40 % of the protein. Thus, the

truncated Ufd1 protein lacks the SHP motif required for interaction with Cdc48, as well as

the SIM motif required for recognizing SUMO conjugates. It is surprising that loss of

Cdc48 interaction module in Ufd1 does not affect cell viability, suggesting that either the

essential function of Ufd1 is not assigned to association with the hexameric ring or that

Ufd1 might establish interaction with Cdc48 bridged by other proteins like Npl4.

Interestingly, a screen for fission yeast mutants resistant to the growth inhibitor

cordycepin has previously identified the same frame shift mutation in ufd1, caused by

insertion of a cytosine in front of an ATC codon for isoleucine 210 (Naula et al., 2003),

suggesting that this particular position in the S. pombe genome might be prone to

mutations. Assuming that all the isolated intragenic suppressors confer structural

stabilization to the Cdc48-353 ring, it is possible that Ufd1 truncation affects the stability

of the Cdc48-353 in a similar fashion. The C-terminal region of Ufd1 is predicted to be

intrinsically disordered and loss of this region might contribute to increased structural

stability of the Cdc48-353 complex.

In this study we describe a simple and cost effective strategy for identifying single-

site variants by genome wide sequencing in Schizosaccharomyces pombe. Moreover, this

approach allows the identification of mutations that do not give rise to any dramatic

phenotype, as it is the case with cdc48-353sup

-29. The initial design of our screen involved

selection of mutants that had also acquired a cs phenotype. This would have been useful

for subsequent identification of the mutated genes by complementation. However, this

approach appears to restrict the final results of the study resulting in the selection of

mainly intragenic suppressors. It is possible that further analyses of suppressor mutants

which to do exhibit a cs phenotype would reveal more extragenic suppressors in essential

or non-essential genes. Our results suggest that structural stability of the functional Cdc48

complex requires tight inter-subunit communication. Perturbations of subunit-subunit

contacts may result in loss of Cdc48/p97 chaperone functions, as it is the case with some

human neurological diseases which are due to single amino acid substitutions in p97

(Tang and Xia, 2013)


41

Materials and Methods

Yeast strains, media and general genetic methods

Standard genetic techniques and growth media for S. pombe were applied as described

previously (Moreno et al., 1991; Forsburg and Rhind, 2006). S. pombe strains used in this

study are listed in Table 2. Double mutants were obtained by genetic crosses and random

spore analysis.

Strains expressing GFP-Suc22 and Spd1-S43A/S43D were constructed as follows.

pJ201 vectors (pON1140 and pON1141) containing the ORF of spd1 with the desired

mutations (S43A and S43D respectively) flanked by 130 nt of the genomic upstream and

downstream regions of spd1 were produced by DNA2.0. Mutant spd1 constructs were

amplified by PCR with plasmids pON1140 and pON1141 as templates and the primer set

Spd1F and Spd1 R (5’-TCCGTGTTCGATAGGCATTC-3’ and 5’-TAATGGTATAGG

GAATCAAC-3’). Each PCR product was used for transformation of Eg3340 using

cryopreserved competent cells (Suga and Hatakeyama, 2005). 5-FOA resistant clones

were selected. Correct genome integration of the mutant alleles was confirmed by PCR

with primer set Spd1+207 (5’-TAACAAGGAGAGACAAATGC-3’) and spd1-304 (5’-

GAGAAAGCGAATTTAATTGC-3’). The fidelity of the integrated construct was verified

by DNA sequencing.

Strains Eg3751 and Eg3752 containing the construct spd1-S43D-VC155::natMX6

were generated as follows. Wild type spd1-VC155::natMX6 was amplified by PCR with

genomic DNA from Eg3653 as a template and primer set Spd1F and Spd1R and the PCR

product was cloned into TOPO Blunt vector (Invitrogen). The vector with the integrated

construct was used as a template for PCR site-directed mutagenesis with primer set Spd1-

S43D-F (5’-GGAATGCGGGTTCGTAAAGATATTTCCACCGGATACAAG-3’) and

Spd1-S43D-R (5’-CTTGTATCCGGTGGAAATATCTTTACGAACCCGCATTCC -3’),

followed by digestion with DpnI to produce plasmid pON1170. pON1170 was used as a

templated in a PCR reaction with primer set Spd1F and Spd1R, and the PCR product was

used for transformation of Eg3340. Colonies were selected for 5-FOA and NAT

resistance.

Strains Eg3753 and Eg3754 expressing spd1-S43L-VC155::natMX6 were constructed

like Eg3751 and Eg3752 with primer set introducing the S43L mutation Spd1-S43L-F (5’-


42

GGAATGCGGGTTCGTAAATTAATTTCCACCGGATACAAG-3’) and Spd1-S43L-R

(5’-CTTGTATCCGGTGGAAATTAATTTACGAACCCGCATTCC-3’) to produce

plasmid pON1171.

Strains from Eg3985 to Eg3991 were constructed as follows. VC155:natMX6

construct was amplified by PCR with purified genomic DNA from Eg3607 as a template

and the primer set Spd1F-w/o-stop-codonF and Spd1R (5’-GAGGGTACCA

ATGAGAGATTA and 5’-TAATGGTATAGGGAATCAAC-3’). The spd1 locus

containing the desired mutations was amplified by PCR with purified genomic DNA as a

template from strains Eg3355 (S43A) and Eg3513 (V40G) and the primer set Spd1F and

Spd1-w/o-stop-codonR (5’-TCCGTGTTCGATAGGCATTC-3’ and 5’-TAATCTCTCAT

TGGTACCCTC -3’). The two PCR products containing the VC155:natMX6 construct and

the spd1-S43A or spd1-V40G constructs were used for a fusion PCR with primers Spd1F

and Spd1R. The resulting amplicon was used for transformation of Eg3340. NAT-resistant

clones were selected, positive clones were tested for correct genome integration by PCR,

and the fidelity of the integrated construct was verified by DNA sequencing

(Eurofinsgenomics).

Table 2. S. pombe strains used in this study

Strain Genotype Source

Eg 282 h90

Laboratory

stock

Eg 325 h90

ura4-D18 Laboratory

stock

Eg 337 h90

leu1 Laboratory

stock

Eg 544 h- d-mat2/3::LEU2

+

Laboratory

stock

Eg 545 h+ d-mat2/3::LEU2

+

Laboratory

stock

Eg 1407 h90

ddb1::kanMX6 spd1::ura4+ ura4-D18

Laboratory

stock

Eg 1410 h- spd1::hphMX6 ura4-D18 leu1 ade6-704

Laboratory

stock

Eg 1420 h+ d-mat2/3::LEU2

+ spd1::hphMX6 ura4-D18

Laboratory

stock


43

Eg 2193 h- ddb1::natMX6 spd1::ura

+ ura4-D18

Laboratory

stock

Eg 2352 h- leu1-32 ura4-D18

Laboratory

stock

Eg 2358 h90

Δddb1::kanMX6 Δspd1::hphMX6 leu1-32 ura4-D18 Laboratory

stock

Eg 2447 h+ ddb1::natMX6

Laboratory

stock

Eg 2634 h+ rad3-ts ura4-D18 leu1-32 ade6-704

Laboratory

stock

Eg 2735 h+ ddb1::natMX6 spd1::ura4

+ ura4-D18 leu1-32 rad3-ts

Laboratory

stock

Eg 2736 h- ddb1::natMX6 rad3-ts

Laboratory

stock

Eg 2738 h- ddb1::nat spd1::ura4+ ura4-D18 rad3-ts

Laboratory

stock

Eg 2750 h+ ddb1::kanMX6 spd1::hphMX6 GFP-suc22 leu1

Laboratory

stock

Eg 2827 h- rad3-ts spd1::hphMX6

Laboratory

stock

Eg 3026 h+ 2,3::leu1

+ rad3-ts spd1::hphMX6 leu1-32

Laboratory

stock

Eg 3156 h-S

B. Grallert

Eg 3219 h90

Δddb1::kanMX6 ura4-D18 Laboratory

stock

Eg 3340 h- Δspd1::ura4

+ GFP-suc22 ade6-704 leu1-32 ura4-D18 A.M. Carr

Eg 3349 h- spd1-S43D GFP-suc22 ade6-704 leu1-32 ura4-D18 This study

Eg 3355 h- spd1-S43A GFP-suc22 ade6-704 leu1-32 ura4-D18 This study

Eg 3445 h90

Δddb1::natMX6 spd1-S43D GFP-suc22 ura4-D18 This study

Eg 3446 h90

Δddb1::natMX6 spd1-S43A GFP-suc22 leu1-32 This study

Eg 3461 h+ Δddb1::natMX6 spd1-S43D leu1-32 ura4-D18 ade6-704 This study

Eg 3462 h- Δddb1::natMX6 spd1-S43D GFP-suc22 leu1 ura4-D18 This study

Eg 3463 h- Δddb1::natMX6 spd1-S43D rad3-ts leu1 ura4-D18 ade6-704 This study

Eg 3464 h+Δddb1::natMX6 spd1-S43D rad3-ts leu1 ura4-D18 This study

Eg 3467 h+Δddb1::natMX6 spd1-S43A ade6-704 leu1 ura4-D18 This study

Eg 3468 h- Δddb1::natMX6 spd1-S43A leu1-32 ura4-D18 This study

Eg 3476 h90

Δddb1::kanMX6 spd1-S43A ade6-704 ura4-D18 This study


44

Eg 3477 h90

Δddb1::kanMX6 spd1-S43A ade6-704 ura4-D18 leu1-32 This study

Eg 3478 h90

Δddb1::kanMX6 spd1-S43D ade6-704 ura4-D18 This study

Eg 3481 h- spd1-S43D rad3-ts leu1-32 This study

Eg 3482 h- spd1-S43A rad3-ts leu1-32 ade6-704 This study

Eg 3483 h+ spd1-S43A rad3-ts leu1-32 ade6-704 ura4-D18 This study

Eg 3512 h- spd1-V40G leu1-32 This study

Eg 3513 h+ spd1-V40G leu1-32 ade6-704 This study

Eg 3514 h - Δcdt2::kanMX6 spd1-S43L leu1-32 ura4-D18 This study

Eg 3538 h- Δddb1::nat spd1-S43A ura4-D18 ade6-704 rad3-TS This study

Eg 3575 h+ spd1-S43D ura4-D18 leu1-32 This study

Eg 3597 h? gad8-K259R ura4-D18 J. Petersen

Eg 3607 h

+ VN173-pcn1::kanMX6 spd1-VC155::natMX6 leu1-32 ade6-

M216 ura4-D18 S. Kersey

Eg 3653

h+ leu1

+::nmt41-cdt2 Δcdt2::ura4

+ ura4::adh-dmdNK-

natMX6-adh-hENT VN173-pcn1::kanMX6 spd1-

VC155::natMX6

This study

Eg 3684 h+ spd1-S43A leu1-32 ura4-D18 This study

Eg 3751 h

- spd1-S43D-VC155-natMX6 GFP-suc22 ade6-704 leu1-32

ura4-D18 This study

Eg 3752 h

- spd1-S43D-VC155-natMX6 GFP-suc22 ade6-704 leu1-32

ura4-D18 This study

Eg 3753 h

- spd1-S43L-VC155-natMX6 GFP-suc22 ade6-704 leu1-32

ura4-D18 This study

Eg 3754 h

- spd1-S43L-VC155-natMX6 GFP-suc22 ade6-704 leu1-32

ura4-D18 This study

Eg 3793 h+ ∆ddb1::natMX6 spd1-S43L ura4-D18 leu1-32 This study

Eg 3794 h+ ∆ddb1::natMX6 spd1-S43L ura4-D18 leu1-32 This study

Eg 3795 h- ∆ddb1::natMX6 spd1-S43L rad3-ts ura4-D18 leu1-32 This study

Eg 3796 h- ∆ddb1::natMX6 spd1-S43L rad3-ts ura4-D18 leu1-32 This study

Eg 3797 h- ∆ddb1::natMX6 spd1-V40G ura4-D18 leu1-32 This study

Eg 3798 h+ ∆ddb1::natMX6 spd1-V40G rad3-ts ura4-D18 leu1-32 This study

Eg 3799 h+ ∆ddb1::natMX6 spd1-V40G ura4-D18 leu1-32 This study


45

Eg 3800 h90

∆ddb1::kanMX6 spd1-V40G ura4-D18 This study

Eg 3801 h- spd1-S43L rad3-ts leu1-32 ura4-D18 This study

Eg 3802 h+ spd1-S43L rad3-ts leu1-32 ura4-D18 This study

Eg 3822 h- spd1-V40G rad3-ts This study

Eg 3823 h+ spd1-V40G rad3-ts leu1-32 ura4-D18 This study

Eg 3824 h- spd1-S43L ura4-D18 leu1-32 This study

Eg 3825 h+ spd1-S43L ura4-D18 This study

Eg 3826 h

- VN173-pcn1:kanMX6 spd1-S43L-VC155:natMX6

leu1+::nmt41-cdt2 ∆cdt2::ura4

+

This study

Eg 3827

h+ VN173-pcn1:kanMX6 spd1-S43L-VC155:natMX6


+ ura4::adh-dmdNK-natMX6-

adh-hENT

This study

Eg 3828 h

+ VN173-pcn1:kanMX6 spd1-S43D-VC155:natMX6


+

This study

Eg 3829

h+ VN173-pcn1:kanMX6 spd1-S43D-VC155:natMX6


+ ura4::adh-dmdNK-natMX6-

adh-hENT

This study

Eg 3871 h+ spd1-V40G ura4-D18 leu1-32 This study

Eg 3877 h90

Δddb1::kanMX6 spd1-S43L ura4-D18 This study

Eg 3878 h- spd1-V40G ura4-D18 leu1-32 ade6-704 This study

Eg 3879 h- spd1-V40G ura4-D18 leu1-32 This study

Eg 3880 h+ spd1-V40G ura4-D18 leu1-32 ade6-704 This study

Eg 3892 d-mat2/3? leu1-32 spd1::hphMX6 gad8-K259R J. Petersen

Eg 3893 d-mat2/3? leu1-32 spd1::hphMX6 gad8-K259R J. Petersen

Eg 3945 h

+ leu1


+ adh-suc22::ura4-D18

VN173-pcn1::kanMX6 spd1-VC155::natMX6 This study

Eg 3946 h

+ leu1


+ adh-suc22::ura4-D18

VN173-pcn1::kanMX6 spd1-VC155::natMX6 This study

Eg 3947 h

+ leu1


+ VN173-pcn1::kanMX6

spd1-VC155::natMX6 This study

Eg 3948 h

+ leu1


+ VN173-pcn1::kanMX6

spd1-VC155::natMX6 This study

Eg 3949 h+ spd1-V40G GFP-suc22 ura4-D18 leu1-32 This study


46

Eg 3950 h- spd1-V40G GFP-suc22 ura4-D18 leu1-32 ade6-704 This study

Eg 3951 h- spd1-S43L GFP-suc22 ura4-D18 ade6-704 This study

Eg 3952 h+ spd1-S43L GFP-suc22 ura4-D18 This study

Eg 3963 h- spd1-S43L GFP-suc22 ura4-D18 ade6-704 leu1-32 This study

Eg 3970 h

- spd1-S43A-VC155::natMX6 GFP-suc22 ade6-704 leu1-32

ura4-D18 This study

Eg 3971 h

- spd1-S43A-VC155::natMX6 GFP-suc22 ade6-704 leu1-32

ura4-D18 This study

Eg 3972 h

- spd1-V40G-VC155::natMX6 GFP-suc22 ade6-704 leu1-32

ura4-D18 This study

Eg 3985 h

+ leu1


+ VN173-pcn1:kanMX6

spd1-S43A-VC155:natMX6 This study

Eg 3986 h

- leu1


+ VN173-pcn1:kanMX6 spd1-

S43A-VC155:natMX6 This study

Eg 3987 h

+ leu1


+ VN173-pcn1:kanMX6

spd1-S43A-VC155:natMX6 This study

Eg 3988 h

+ leu1


+ VN173-pcn1:kanMX6

spd1-V40G-VC155:natMX6 This study

Eg 3989 h

+ leu1


+ VN173-pcn1:kanMX6


Eg 3990 h

+ leu1


+ VN173-pcn1:kanMX6


Eg 3991 h

+ leu1


+ VN173-pcn1:kanMX6


OL 1491 h- Δddb1::kanMX6 GFP-suc22

Laboratory

stock

OL 1696 h- GFP-suc22 ura4-D18

Laboratory

stock

Strains from Rasmus Hartmann-Petersen collection

1235 cdc48-G338D/P481R This study

1237 cdc48-G338D/S359G/P481R This study

1239 cdc48-G338D/L524F/K715N This study


1243 cdc48-G338D/R369P This study


47

1244 cdc48-G338D/Q781stop This study

1245 cdc48-G338D/Q781stop This study

1246 cdc48-G338D/E598V This study

1247 cdc48-G338D/E598V This study

1248 cdc48-G338D/P292S This study

1250 cdc48-G338D/P481A This study

1251 cdc48-G338D/P481E This study


1257 cdc48-G338D/R644H This study


1265 cdc48-G338D/R644H This study


1271 cdc48-G338D/P292T This study

1275 cdc48-G338D/R764S This study


1285 cdc48-G338D/P516L This study

1286 cdc48-G338D/E325K This study

1289 cdc48-G338D/V767A This study

1291 cdc48-G338D/N371S This study

Sensitivity assays

Strains were grown to stationary phase in YEL at 30 ºC or 25 ºC when temperature

sensitive mutants were tested. Cell titers were adjusted to 107 cells/ml and tenfold serial

dilutions were prepared. 5 µl of each dilution were spotted onto YEA plates without drugs

or on plates containing HU, MMS or zeocin. Plates were cast 2 days prior to use and kept

at room temperature. Pictures were taken after 3 days of incubation at 30 ºC (or at

indicated temperature).


48

Protein extraction and Western blotting

To study protein levels in response to hydroxyurea treatment, cultures were grown to

mid-log phase at 30 ºC in YE and 1x107 cells were harvested. 20 mM hydroxyurea was

added to the remainder of the cultures and 1x107 cells were harvested at the indicated time

points. Protein extracts for Western blotting were prepared by precipitation with TCA

(Caspari et al., 2000). Samples were run on 15 % SDS-PAGE gels and blotted to a mixed

cellulose ester membrane (Advantec) in semidry transfer buffer (24 mM Tris, 192 mM

glycine, 20 % methanol) for 1 hour. The membrane was blocked in 0.05 % PBS-T and 5

% low-fat milk for 1 hour at room temperature and incubated with primary antibody

overnight at 4 ºC. Membrane was washed incubated with secondary antibody for 1 hour at

room temperature and developed with ECL Western blotting detection reagents following

manufacturer instructions (Amersham). Spd1 was detected using polyclonal rabbit anti-

Spd1 antibodies (1:1000 dilution) raised against E. coli purified proteins by Yorkshire

Bioscience. For loading control monoclonal mouse α-TAT antibody was used diluted

1:10000. HRP-conjugated secondary antibodies, rabbit anti-mouse (Dako P0161) and

swine anti-rabbit (Dako P0217) were used in 1:5000 dilution.

Purification of genomic DNA

Genomic DNA of selected strains was purified by phenol/chloroform extraction. Cells

from 100 mL overnight cultures were harvested by centrifugation (3000 g, 5 minutes) and

resuspended in 50 mL SP1 buffer (1.2 M sorbitol, 50 mM sodium citrate, 50 mM sodium

phosphate, 40 mM EDTA, pH 5.6) and pelleted again by centrifugation (3000 g, 5

minutes). Cells were resuspended into 10 ml sterile SP2 buffer (1.2 M sorbitol, 50 mM

sodium citrate, 50 mM sodium phosphate, pH 5.6) containing 2 mg/mL glucanex

(Novozymes). After incubation for 45 minutes at 37 °C, protoplasts were isolated by

centrifugation (3000 g, 5 minutes) and resuspended in 7.5 mL 5 x TE buffer (10 mM

Tris/HCl, 1 mMEDTA, pH 8.0). The protoplasts were lysed by adding 0.75 mL of 10 %

(w/v) SDS and incubating for 5 minutes at RT. Then 2.5 ml 5 M potassium acetate was

added and the mixture was incubated on ice for 30 minutes, before centrifugation at 5000

rpm for 15 minutes. The entire supernatant was transferred to a fresh tube before addition

of 10 mL of cold isopropanol. After centrifugation at 8000 rpm for 15 minutes, the

precipitate was washed three times with 70% (v/v) ethanol and dried in a vacuum


49

centrifuge at 30 °C. The precipitate was then resuspended in 500 μL TE buffer containing

20 μg/mL RNase A (Sigma) and incubated at 37°C for 1 hour. RNase was deactivated by

adding 15 μl 10% SDS and 5 μl 5mg/ml Proteinase K, followed by incubation at 50 ºC for

30 minutes. DNA was extracted four times with phenol/chloroform

phenol:chloroform:isoamylalchohol 25:24:1 (Sigma) and once with chloroform (Sigma).

Finally, the DNA was precipitated by adding 1/10 volume 5 M potassium acetate pH 5.2

and 2.5 volumes 96 % ethanol and resuspended into 200 μL of TE buffer.

Live cell imaging

All microscopy was conducted with live (unfixed) cells.

Imaging was performed at 25 °C using a DeltaVision Imaging System (GE

Healthcare) comprised of an Olympus IX-71 inverted wide-field microscope, an Evolve

EMCCD camera, and an Insight solid-state illumination unit. Stacks of z-series were

acquired with 0.5 μm step size and deconvolved in SoftWoRx software (Applied

Precision). Images were processed using Volocity software (Perkin Elmer). GFP-Suc22

was expressed from its endogenous locus and cells were grown at 30 ºC in YEL.

Logarithmically growing cells were analyzed before and after treatment with 20mM HU

for 2h. In BiFC experiments VN173-pcn1 and spd1-VC155 were expressed from their

native promoters. Cells were grown at 30ºC in filter-sterilized MSL medium in absence or

presence of thiamine (5 µg/mL).

The constitutively active ADH promoter was used for overexpression of suc22. Cells

overexpressing suc22 were visualized on an AxioImager (Carl Zeiss MicroImaging)

microscope using filter 41017 from Croma. The light source was an HXP 120 metal halide

lamp (Carl Zeiss MicroImaging). Pictures were processed with Volocity software

(PerkinElmer)


50

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Paper I

Single Site Suppressors of a Fission Yeast

Temperature-Sensitive Mutant in cdc48

Identified by Whole Genome Sequencing

Irina N. Marinova, Jacob Engelbrecht, Adrian Ewald, Lasse L. Langholm,

Christian Holmberg, Birthe B. Kragelund, Colin Gordon, Olaf Nielsen,

Rasmus Hartmann-Petersen

PLoS ONE 2015, doi:10.1371/journal.pone.0117779

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Documents

Temperature Sensitive Mutants in Fission Yeast Marinova.pdfSingle Site Suppressors of a Fission Yeast Temperature-Sensitive Mutant in cdc48 Identified by Whole Genome Sequencing Irina