Roles for RecQ Helicases inTelomere Preservation

Patricia L. Opresko

University of Pittsburgh Department of Environmental and Occupational HealthBridgeside Point100 Technology Drive, Suite 350Pittsburgh, PA 15219-3130plo4@pitt.edu

Werner Syndrome

14 Years Old

48 Years Old

Symptoms Average Age of Onset (yrs)Greying of hair 20Wrinkling of the skin 25.3Loss of hair 25.8Cataracts 30Skin Ulcers 30Diabetes (type II) 34.2Death 47

OsteoporosisAtherosclerosisCancer

RecQ, E. coli

Sgs1, S. cer.

Rqh1, S. pombe

RecQL, H. sapiens

BLM, H. sapiens

WRN, H. sapiens

RecQ4, H. sapiens

RecQ5β, H. sapiens

FFA-1, X. laevis

RecQ5β, D. melanogaster

Helicase3’ to 5’

Exonuclease3’ to 5’

Acidic NLSRecQconserved

HRDC

RecQ Family “Care Takers” of the Genome

Cellular defects in WS cell lines

• Mitotic Homologous DNA Recombination– Defect in resolving

intermediates

• Genomic Instability– Chromosomal

rearrangements,translocations, dicentrics

– Large deletions

• DNA Repair– Hypersensitivity to

4-NQODNA crosslinking agentstopoisomerase inhibitorsmethyl methanesulfonate

• Replication– Reduced replicative lifespan– Extended S-phase • Telomere instability

Telomere-Associated Replicative Senescence

Telomerase

ALT

senescence

Germ

Somatic

+ exogenoustelomerase

telomere-dependent

Modified from Campisi 2001 Exp. Geron.

Adult stem

Germ cells:sufficient telomerase activity- no shortening

Adult stem cells: variable levels of telomerase activity- slow shortening

Somatic cells:most have no telomerase activity- exhibit faster rates of shortening

Cancer cells:90% show high telomerase activity10% use an alternative pathway- no telomere loss

senescence

Loss of telomeric

DNAproteins

Activate DNA damage response

Senescence/ Apoptosis

3’

CLOSED

(TTAGGG)n

(AATCCC)n

Telomeres Protect Chromosome EndsComplex of Protein and DNA

TRF1TRF2

POT1

Bind duplex repeats

Binds single strand DNATTAGGGTTAG

Genomic instability

DNA RepairALT (HR)

Telomerase

OPEN

(AATCCC)n

(TTAGGG)n3’

Shelterin/ 6 protein complex

Telomere Dysfunction Contributes to WS Pathology

Exhibit telomere loss1. Accelerated decrease of mean telomere lengths (Shulz 1996)2. Increased loss of telomeres from sister chromatids

(Crabbe 2004)

Expression of either WRN or telomerase can prevent1. Premature senescence (Wyllie 2000)2. Sister telomere loss (Crabbe 2004)3. Accumulation of aberrant chromosomes (fusions, breaks, translocations)

(Crabbe 2007)

Wrn-/- mice appear normal

late generation Wrn-/-Terc-/- mice with shortened telomeres exhibit WS phenotypes (Chang 2004, Du 2004)

WS primary fibroblasts

Mouse models

Telomere Replication

Evidence For WRN Activity at Telomeres

HcRed-PCNAECFP-TRF1EYFP-WRN

WRN localizes to telomeres in S-phase telomerase deficient cells • In telomerase-negative ALT cells (Opresko 2004)

• In primary fibroblasts- WRN helicase prevents the loss of telomeres replicated from the G-rich

lagging strand; by CO-FISH (Crabbe 2004).- Pot1a and FEN1 defects also cause preferential loss of lagging

strand telomeres (Wu 2006; Saharia 2008)

3’

Evidence For WRN Activity at Telomeres

WRN and POT1 suppress aberrant telomere recombination and exchanges(Laud 2005, Wu 2006, He 2006, Li 2008)

Late generation Wrn-/-Terc-/- mice and Pot1a-/- mice exhibit:

increased telomeric sister chromatid exchangesintra-telomere recombination

WS human and Pot1a -/- mouse cells exhibit

increased telomere circlesHJ cleavage of telomere T-loop

Telomere Recombination

Griffith et al 1999, Cell

WRN and BLM Roles at Telomeres

Telomere BindingProteins

TRF1 POT1TRF2

WRN BLM

intra- or inter-telomericG-quadruplex

3’

Hypothesis: WRN and BLM protein cooperate with telomeric proteins to dissociate alternate DNA structures at telomeres during replication and repair

• TRF2 recruits WRN and BLM to telomeric DNA (Opresko 2002; Machwe 2004)• POT1 physically binds WRN in HeLa cells (BLM interaction is weaker)

(Opresko 2005)

3’3’5’

5’3’

3’

5’3’

Inter-telomeric D-loopIntra-telomeric D-loop

POT1 stimulates the WRN and BLM helicase activity

X-WRN - + + + + - RecQ - + + + + +POT1 - - ▲ + POT1 - - ▲

Opresko 2005

X-WRN - + + + + POT1 - - ▲ +

0 2 4 6 8 10 12 14 16 1805

1015202530354045

% D

ispl

acem

ent

Time (min.)

+POT1POT1• Increases the amount and rate of WRN strand

displacement; also BLM helicase

• Does not alter WRN or BLM unwinding of a non-telomeric fork

• Does not alter unwinding by bacterial helicases UvrD and RecQ

(TTAGGG)45’ 5’34 bp

34 bp

3’

5’ 5’

3’

5’

3’

▪ WRN exonuclease is inefficient on short ssDNA

▪ WRN is inactive on blunt ended duplex DNA

▪ Junctions in the substrate active the exonuclease at blunt ends

WRN Helicase and Exonuclease Cooperate to Dissociate Fork-like Substrates

exohelicase

5’3’

exonucleasehelicase

▪ Also cooperate to release invading strand of a D-loop

X

WRN - + + + + + + POT1 - - R Δ

D-loop

shortenedproducts

(TTAGGG)4

shortenedproducts

WRN - + + + + + + + + POT1 - - -

ATP + + - + + + - - -

• POT1 does not alter the WRN exonuclease in the absence of ATP/helicase activity

Native Gel

(Opresko, JBC 2005)

(TTAGGG)4

34 bp

POT1 Limits WRN Exonuclease by Stimulating WRN Helicase

2nd Binding cycle

1st Binding cycle

Possible Mechanisms of WRN Helicase Stimulation by POT1

POT1

WRN helicase

WRN exo

X

X

5’3’

(TTAGGG)4

> 39 nt

< 33 nt

X

> 39 nt

%TD 71 71 71 69

WRN - + + + + POT1 - -

Sho

rter

Pro

duct

sCan POT1 Pre-loading Stimulate WRN Helicase ?

(TTAGGG)4

34 bp3’

POT1 pre-loading - is not sufficient to stimulate WRN helicase- does not prevent WRN activity

WRN - + + + + POT1 - -

% TD 89 89 88 73

WRN - + + + + POT1 - -

(TTAGGG)4

22 bp3’(TTAGGG)4

22 bp3’

%TD 66 63 61 51

WRN - + + + + - - - + + + -- - + - - +

shortenedproducts

POT1 and RPA Differ in Regulation of WRN onOpen Telomeric Ends

POT1WRN helicase

WRN exo

POT1

SSBP

RPA

6x↓ 19x↑

3’36 bp

(TTAGGG)7

POT1 inhibits WRN, RPA stimulates

WRN - + + + +POT1 - -

Non-telomeric tail

3’36 bp

(TTAGGG)3TTAG

POT1 inhibition requires a telomeric tail

Addition of a 5’ ssDNA Tail (Fork) RestoresPOT1 Stimulation of WRN

3’

36 bp

(TTAGGG)7

WRN - + + + + -POT1 - - +

Shorter

Products

- + + + + - -

Telomeric Tail

exo helicase

+ATP +ATPγS (no helicase activity)

GGTTAGGGTTAGGGTTAG

3’ tail sequence

GGTTACGGTTAGGGTTAGGG

GGTTAGGGTTAGGCTTAGGG

CTGTTTGCATCGATCTGCMix

Tel

Tel-A

Tel-B

Tel-G

GGTTAGGGTTAGGGTTAGGG

1.5

3.7

1.8

2.3

2.6

Fold Increase

Inta

ct T

elom

eric

Pro

duct

POT1 (nM)

Tel

MixTel-A

Tel-B

0 20 40 60 80 100 120

2

4

Tel-G3

1

POT1 Pre-loading Promotes WRN HelicaseUnwinding of Telomeric Forks

= POT1 binding site

POT1 loading near the junction (Tel-B) is more important for WRN stimulation

3’

(TTAGGG)3 TTAG

Stimulation

Inhibition of activity

No effect

Stimulation

Effect on WRN Helicase

Substrate

Summary of POT1 Modulation of WRN Activity

3’

3’

3’

3’

=(TTAGGG)nExonuclease notaltered directly

POT1 binding mode may regulate WRN activity

3’

OFF

POT1 WRN

ON

3’POT1

WRNATC-5’

Kibe et al MBC, 2007, p. 2378.Fission Yeast Taz1 and RPA Are Synergistically Required to Prevent Rapid Telomere Loss

POT1 May Protect Telomeric Ends fromFraying by DNA Helicases

• Yeast lacking Taz1 (TRF2) and expressing mutant RPA (mRPA)exhibit rapid telomere loss

• Telomere loss is suppressed ifRqh1 (RecQ) is knocked out ORPOT1 is overexpressed

• Coating of the telomeric tail with POT1 vs. RPA has profound consequences for WRN helicaseactivity

WRN - + + + + POT1 - -

POT1 nMR

atio

of l

ong:

shor

tun

wou

nd fo

rks

POT1 Does Not Retain WRN on Telomeric Forks During Unwinding

0.0

1.0

2.0

3.0

4.0

5.0

0 50 100 150

POT1 does not alter the ratio of unwound long telomeric forks to short mixed sequence forks

POT1 bound to ssDNAproduct

3’

3’

Telomeric 34 bp

Mix 22 bp

MCM helicase N-terminus increases the ratio of unwound long:short duplexes- acts as a processivity clamp (Barry et al 2007, NAR)

Summary and Conclusions

1. POT1 stimulates WRN and BLM helicases, but not E. coli RecQ- species specific

2. POT1 pre-loading on telomeric tails:A. is not sufficient to stimulate WRN; does not recruit WRN

B. inhibits WRN activity on 3’ tailed telomeric duplexes- POT1 protects telomeres in the OPEN form

C. stimulates WRN unwinding of telomeric forks- POT1 interaction with the ssDNA/dsDNA junction regulates WRN

3. POT1 does not retain WRN on telomeric forks during unwinding- stimulation is by preventing strand re-annealing rather than WRN

dissociation

4. WRN show increased processivity on plasmid D-loops compared to oligomeric D-loops- POT1 stimulates WRN helicase on telomeres in the CLOSED form

Roles for WRN Protein at Telomeric Ends

Replication stallin telomere

Fork collapse and DS break

Sister telomereloss

Restore replication fork

Initiation of HRmediated repair

T-SCEshortened telomere

- WRN +WRN

- WRN - WRN

+ WRN

Dissociate withoutstrand cross over

Resolve withstrand cross over

telomerase

POT1

TRF2

WRN helicase

WRN exo5’3’

exonucleasehelicase 5’3’ exonucleasehelicase

3’

5’3’

exonucleasehelicase

Acknowledgements• Vilhelm Bohr, NIA• Ming Lei, U. of Michigan• Peter Baumann, Stowers Inst.• James Keck, U. of Wisconsin• Walter Chazin, Vanderbilt

Funding• Ellison Medical Foundation• NIEHS

Opresko lab• Jerry Nora• Greg Sowd• Fujun Liu• Rama Damerla