Brc1-mediated DNA repair and damage tolerance

Daniel M. Sheedy1,2, Dora Dimitrova1, Jessica K. Rankin1, Kirstin L. Bass1, Karen M.

Lee1, Claudia Tapia-Alveal1, Susan H. Harvey1,2, Johanne M. Murray3 and Matthew J.

O’Connell1,2,*

1Department of Oncological Sciences, Mount Sinai School of Medicine, New York New

York 10029

2Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, Melbourne,

Victoria 8006, Australia

3Genome Damage and Stability Centre, School of Biological Science, University of

Sussex, Falmer, Brighton BN1 9RQ United Kingdom.

*Corresponding author:

Department of Oncological Sciences, Mount Sinai School of Medicine, One Gustave L.

Levy Place. New York, NY 10029

Phone: 212-659-5468

Fax: 212-987-2240

Email: matthew.oconnell@mssm.edu

Genetics: Published Articles Ahead of Print, published on June 21, 2005 as 10.1534/genetics.105.044966

Abstract

The structural maintenance of chromosome (SMC) proteins are key elements in

controlling chromosome dynamics. In eukaryotic cells, three essential SMC complexes

have been defined: cohesin, condensin and the Smc5/6 complex. The latter is essential

for DNA damage responses; in its absence both repair and checkpoint responses fail. In

fission yeast, the UV-C and ionizing radiation (IR) sensitivity of a specific hypomorphic

allele encoding the Smc6 subunit, rad18-74 (renamed smc6-74) is suppressed by mild

overexpression of a 6 BRCT-domain protein, Brc1. Deletion of brc1 does not result in a

hypersensitivity to UV-C or IR, and thus the function of Brc1 relative to the Smc5/6

complex has remained unclear. Here we show that brc1∆ cells are hypersensitive to a

range of radiomimetic drugs that share the feature of creating lesions that are an

impediment to the completion of DNA replication. Through a genetic analysis of brc1∆

epistasis and defining genes required for Brc1 to suppress smc6-74, we find that Brc1

functions to promote recombination through a novel post-replication repair pathway and

the structure-specific nucleases Slx1 and Mus81. Activation of this pathway through

overproduction of Brc1 bypasses a repair defect in smc6-74, re-establishing resolution of

lesions by recombination.

Introduction

Chromosomal integrity is essential for cell viability. Not surprisingly therefore, cells have

evolved a plethora of enzymes to control the chromosome dynamics required for

transcription, DNA replication, mitosis and the repair of a wide range of DNA lesions and

stalled replication intermediates. Such events are coordinated with cell cycle

progression, thus ensuring the fidelity of chromosomal inheritance.

The structural maintenance of chromosome (SMC) proteins were identified by

genetic screens in Saccharomyces cerevisiae for chromosome instability mutants.

These chromatin-associated proteins are characterized by N- and C-terminal globular

domains respectively containing Walker A and B ATPase motifs. Two coiled-coil

domains with a central flexible hinge separate these globular domains. Initially, two

highly conserved protein complexes were characterized, termed condensin and cohesin.

Both complexes contain heterodimers of SMC subunits: Smc2 and 4 in condensin, and

Smc1 and 3 in cohesin. Each Smc subunit folds at the hinge to form an intra-molecular

interaction, and are then thought to associate inter-molecularly through the hinge

domain. Each complex contains additional unique non-Smc subunits, and additional

proteins most likely involved in their loading onto chromatin have also been identified. As

their name suggests, condensin functions in mitotic chromosome condensation, and

cohesin in sister chromatid cohesion (HARVEY et al. 2002; HIRANO 2002; HIRANO 2005).

However, mutants in subunits of both complexes have also uncovered DNA repair

defects (AONO et al. 2002; LEHMANN 2005; NAGAO et al. 2004; STROM et al. 2004; UNAL

et al. 2004), most likely through an indirect effect based on a more fundamental defect in

chromosomal dynamics rather than a direct enzymatic role in reversal of a lesion.

A third Smc complex, termed the Smc5/6 complex, is also present in all

eukaryotes (COBBE and HECK 2000; HARVEY et al. 2004). This was originally defined by

the cloning of the Schizosaccharomyces pombe rad18 gene (LEHMANN et al. 1995),

which encodes Smc6 and to clarify the nomenclature, rad18 shall be referred to as

smc6. Together with Smc5 and four non-Smc subunits (Nse1-4, nse4 is identical to

rad62 (MORIKAWA et al. 2004)), these proteins form an essential complex of which the

precise molecular function remains unknown (ANDREWS et al. 2005; FUJIOKA et al. 2002;

HARVEY et al. 2004; HAZBUN et al. 2003; MCDONALD et al. 2003; PEBERNARD et al. 2004;

SERGEANT et al. 2005; ZHAO and BLOBEL 2005). The hypomorphic smc6 alleles, smc6-X

and smc6-74, are defective in the repair of a diverse array of DNA lesions, and in the

case of smc6-74, a further defect in the maintenance of the Chk1-dependent checkpoint

arrest exists, despite normal activation of Chk1 activity (HARVEY et al. 2004; VERKADE et

al. 1999; VERKADE and O'CONNELL 1998). Spore germination experiments with cells

deleted for either smc6 or nse1, and the analysis of additional smc6 alleles have

confirmed that this complex is indeed required to successfully respond to DNA damage

and pass through mitosis. Further, conditional and hypomorphic alleles of nse1, nse2,

nse3 and nse4, together with a conditional allele of rad60, which encodes a protein

involved in Smc5/6 function without being a member of the complex per se (BODDY et al.

2003; MORISHITA et al. 2002), also show defects in DNA damage responses. Recent

data from S. cerevisiae has demonstrated a defect in the segregation of rDNA at mitosis

in temperature sensitive smc5-6 and smc6-9 mutants, leading to DNA damage that can

be partially suppressed in rad52 mutants, suggesting inappropriate recombination at the

repetitive rDNA (TORRES-ROSELL et al. 2005). This may be related to observed epistasis

between various smc6 and nse1-nse4 alleles and deletion of the Rad51 homolog, rhp51

in S. pombe (LEHMANN et al. 1995; MCDONALD et al. 2003; MORIKAWA et al. 2004;

PEBERNARD et al. 2004). Similar epistasis has been seen between smc6-56 and rad52∆

in S. cerevisiae (TORRES-ROSELL et al. 2005), and here the smc6 mutation also results in

defects in MMS-induced interchromosomal and sister chromatid recombination (ONODA

et al. 2004). Presumably, as with condensin and cohesin, the defective DNA damage

responses of mutants in the Smc5/6 complex are a consequence of a more fundamental

defect in chromosome organization.

Brc1 encodes a six BRCT domain protein that was identified as an allele-specific

high-copy suppressor of smc6-74 (VERKADE et al. 1999). Whilst brc1∆ cells are not

hypersensitive to IR or UV-C, and Brc1 is not part of the Smc5/6 complex, several

observations point towards a DNA damage response function for this protein. Firstly,

brc1 becomes essential in strains with compromised Smc6 or Nse4 function, and is also

synthetically lethal with conditional alleles of rad60 and top2 (BODDY et al. 2003;

MORIKAWA et al. 2004; MORISHITA et al. 2002; VERKADE et al. 1999), which encodes a

type II Topoisomerase. Secondly, as with smc6 mutants, brc1∆ cells loose

chromosomes at 200-fold the rate of wildtype cells, comparable to rhp51∆, and brc1∆

cells frequently show cytologically abnormal nuclei (VERKADE et al. 1999). Finally, a

putative homolog in S. cerevisiae, ESC4, has been shown to be required for DNA repair,

particularly during S-phase, where by an unknown mechanism it may facilitate

resumption of DNA replication (ROUSE 2004), and a more distant human relative, PTIP,

has been implicated in chromosome segregation and DNA damage responses (CHO et

al. 2003; JOWSEY et al. 2004). However, the mechanism by which Brc1 bypasses

defects in Smc6 function in S. pombe cells, including those in G2, is unknown and

important to elucidate.

Here we show S. pombe brc1∆ mutants are indeed sensitive to a range of DNA

damaging drugs, which share the feature of generating lesions during S-phase, or

lesions that are an impediment to DNA replication. Genetic epistasis analysis suggests

that Brc1 may function in this response with the structure-specific nuclease

Mus81/Eme1, and with Rhp51. In addition, we investigated which genes are required for

Brc1 to suppress smc6-74. From these experiments we can further place Brc1 function

on a novel arm of post-replication repair, utilizing translesion synthesis polymerases to

bypass alkylated bases during S-phase, and repair through recombination initiated

largely by the Slx1/Slx4 structure-specific nuclease. Similar Slx1/Slx4 stimulated

recombination is used for Brc1 to bypass the UV-C hypersensitivity of smc6-74 in G2

cells, for which it still requires the post-replication repair machinery, but not translesion

synthesis polymerases. Moreover slx1∆, whilst not hypersensitive to either MMS or UV-

C, dramatically enhances the sensitivities of smc6-74 but not smc6-X. Together, the data

suggest that smc6-74 cells are specifically defective in recombinational repair, and that

Brc1 can, through Slx1/Slx4 generate an alternative structure that can then be repaired

by Smc5/6-independent recombination. Such a defect in smc6-74 may explain the

checkpoint maintenance defects not seen in smc6-X.

Materials and Methods

Fission yeast genetic methods

All strains used were derivatives of 972h- and 975h+. Standard procedures and media

were used for propagation and genetic manipulation (MORENO et al. 1991). Methods for

UV-C survival assays, transformation, microscopy and FACS have been described

previously (DEN ELZEN and O'CONNELL 2004; HARVEY et al. 2004; O'CONNELL et al. 1997;

VERKADE et al. 1999; VERKADE et al. 2001). New null alleles of slx1, mus81 and slx8

were constructed using targeting constructs in which the entire open reading frames

were replaced by ura4. Successful deletion of these genes was confirmed by Southern

blotting. All other strains have been described, or where kindly provided by Dr A.M. Carr

(Genome Damage and Stability Centre, University of Sussex, U.K.), or Dr H. Shinagawa

(Osaka University, Japan).

Drug sensitivity assays

Assays on agar plates were carried out in YES medium, with plates photographed after

4 days at 30°C, or in the case of temperature sensitive mutants, 5 days at 25°C.

Cultures were grown in appropriately supplemented minimal medium to a density of

4x106 cells/ml, and 5µl plated of 10-fold serial dilutions. For liquid exposure to MMS,

cells were grown to 4x106/ml, and MMS added to 0.05%. Samples were taken at

indicated intervals, and MMS inactivated with 5% sodium thiosulfate. Serial dilutions

were then plated in triplicate on YES, and viable colonies counted after 4 days at 30°C.

Percent survival is normalized to samples taken immediately before MMS addition.

Brc1 suppression of smc6-74 assays

Plasmid borne expression of Brc1 from its own promoter, from the wildtype or attenuated

nmt1 promoter with or without thiamine results in equal suppression of smc6-74,

presumably by passing a threshold level required to bypass smc6-74. For these assays,

expression from the attenuated (pRep41) promoter (referred to as pBrc1) under

repressing conditions (5µM thiamine) was used, and compared to controls containing

pRep41 only (referred to as vector).

Results

Brc1 is required for tolerance of radiomimetic drugs

brc1 was originally identified as an allele-specific high-copy suppressor of the UV-C and

IR hypersensitivity of smc6-74. Somewhat surprisingly, brc1∆ cells did not display a

hypersensitivity to these treatments, though did show a high frequency of chromosome

loss, and synthetic lethality with both smc6-74 and smc6-X, suggesting a role in genome

integrity (VERKADE et al. 1999). We considered that Brc1 might be involved either in

repairing a particular type of lesion that is prevalent in smc6 mutants, or be required at a

specific point in the cell cycle. To examine this possibility, we tested the sensitivity of

brc1∆ cells to a range of radiomimetic drugs: methyl methanesulfonate (MMS), 4-

nitroquinoline-N-oxide (4-NQO), hydroxyurea (HU) and camptothecin (CPT). In each

case brc1∆ cells failed to form colonies, and in liquid culture the cells became elongated

due to checkpoint activation (Fig 1), suggesting the hypersensitivity is due to a defect in

repairing lesions caused by these compounds. Although the lesions caused by these

compounds are diverse, they share a commonality of stalling replication and/or of

damaging DNA during replication. Moreover, a plasmid expressing Brc1 could also

rescue the hypersensitivity of smc6-74 cells to these agents. This rescue was complete

for MMS and CPT, but only partial for 4-NQO and HU. From these data we propose that

Brc1 participates in the cellular response to replication damage, and that in smc6-74,

similar structures to those forming in cells treated with these drugs occur or accumulate

following defective DNA repair in response to UV-C and IR, which can then be

processed in a manner mediated by Brc1.

Epistasis analysis of brc1∆

We next used epistasis analysis using the MMS hypersensitivity of brc1∆. In these

experiments, double mutants between brc1∆ and alleles of genes known to function in

various DNA repair pathways were constructed, and their sensitivity to MMS assayed

relative to wildtype and parental controls. MMS sensitivity was assayed by chronic

exposure to a range of concentrations in agar plates, and also by acute exposure to

0.05% MMS in liquid culture, and viability measured after inactivation of the MMS. In

each case both assays gave the same qualitative result. The classic interpretation of

such experiments is that if double mutants have an enhanced hypersensitivity, then

these genes function in different, although potentially overlapping pathways.

Alternatively, if the double mutant is as sensitive as the most sensitive parent, then the

genes function in the same pathway.

These assays employed null and conditional alleles of the following genes to

represent different DNA damage responses: nucleotide excision repair: rad13, rad16

(CARR et al. 1994; CARR et al. 1993); UV-excision repair: rad2, uve1 (MURRAY et al.

1994; BOWMAN et al. 1994); homologous recombination: rhp51, rhp54, rhp57, swi5

(AKAMATSU et al. 2003; JANG et al. 1994; MURIS et al. 1996; TSUTSUI et al. 2000);

double-stranded break repair, intra-S-phase checkpoint: rad32 (TAVASSOLI et al. 1995);

post-replication repair: rhp18, mms2, ubc13, eso1, rev3 and dinB (BROWN et al. 2002;

KAI and WANG 2003; TANAKA et al. 2000; VERKADE et al. 2001); structure-specific

nucleases - replication restart: mus81, slx1, cds1, rqh1, srs2 (BODDY et al. 2000;

COULON et al. 2004; MURAKAMI and OKAYAMA 1995; STEWART et al. 1997; WANG et al.

2001); and checkpoint arrest: chk1, rad3, hus1, rad1 (BENTLEY et al. 1996; CASPARI et

al. 2000; KOSTRUB et al. 1998; WALWORTH et al. 1993). Epistatic interactions were found

only with rhp51∆ and mus81∆ strains (Fig2A, B), suggesting that these genes function in

the same pathway as brc1. However, it is notable that these are among the most MMS

hypersensitive strains. Only brc1∆ rqh1∆ and brc1∆ rhp54∆ double mutants showed

reduced growth compared to parental strains in the absence of MMS (Fig 2D and data

not shown), and brc1∆ is synthetically lethal with smc6-X, smc6-74, top2-191, rad60-1

and rad62-1 (nse4-1) (BODDY et al. 2003; MORIKAWA et al. 2004; MORISHITA et al. 2002;

VERKADE et al. 1999). In all other cases the double mutants were substantially more

sensitive to MMS than either parent (for examples, see Fig 2C, D).

Brc1-mediated rescue of smc6-74: pathways required for MMS tolerance

The above data suggest that Brc1 functions in a pathway that includes Rhp51

and Mus81, but does not rule out that other genes in these experiments are also

involved in a Brc1-mediated response to DNA alkylation, but play additional roles that

are independent of Brc1, and so epistasis is not observed. We therefore sought to

identify the genes required for the Brc1-mediated suppression of the MMS

hypersensitivity of smc6-74. In these experiments, double mutants were made between

smc6-74 and mutants in defined DNA damage response pathways. In cases where

these double mutants were more sensitive than either parent, a criterion that excluded

rhp51, rhp54 and rad2 that are epistatic with smc6-74, cells were transformed with a

Brc1 expressing plasmid or vector only controls, and chronic and acute MMS sensitivity

assayed. A summary of these experiments is presented in Table 1.

We first investigated the post-replication repair pathway, which enables tolerance

of alkylated bases allowing continued DNA synthesis. This is achieved by either lesion

bypass by translesion synthesis polymerases, or by mechanisms involving template

switching by the recombination pathway. Under current models (PRAKASH et al. 2004),

Rhp18 (homolog of S. cerevisiae RAD18, an E3 ubiquitin ligase), together with Rhp6

(homolog of S. cerevisiae RAD6, an E2 ubiquitin conjugating enzyme) catalyzes the

mono-ubiqutination of PCNA. As a result, the translesion synthesis polymerases,

including polymerases η, ζ, and κ (encoded by eso1, rev3 and dinB in S. pombe),

replicate past the lesion for several nucleotides, and then replication continues with the

replicative polymerases such as polymerase δ. However, the monoubiqitinated PCNA

can be polyubuitinated using Lysine-63 linkages by a complex of Ubc13 (an E2),

Mms2/Rad5 (an E3, the latter encoded by rad8 in S. pombe). This signals for bypass by

recombination dependent template switching.

These assays clearly showed that rhp18, but not ubc13, is required for Brc1 to

suppress smc6-74 (Fig 3). Under the model above, this result predicts that the

translesion synthesis polymerases would also be required, and Fig 4A shows this is

indeed the case. A lack of suppression was observed in cells lacking eso1, rev3 and

dinB (denoted 3TLS∆), and a relatively weak suppression in dinB strains. All other

combinations of single and double mutants corroborated these findings (data not

shown), demonstrating that, at least in part, the suppression of the MMS hypersensitivity

of smc6-74 by Brc1 involves this mechanism to bypass stalled replication.

Recent data suggest a role for two checkpoint complexes in the loading of dinB:

the PCNA-related 9-1-1 complex, composed of Rad9, Rad1 and Hus1, and its clamp

loader, composed of Rad17 and the four small subunits of RFC (KAI and WANG 2003). In

keeping with this model, we found little suppression in rad9∆ or rad17∆ backgrounds

(Fig 4B). This lack of suppression was not a consequence of checkpoint failure,

however, as complete suppression was observed in a chk1∆ background (Fig 4C). The

lack of suppression was greater than that seen for dinB∆, suggesting the 9-1-1 and

Rad17/Rfc2-5 may play additional roles in the Brc1-mediated suppression.

Brc1-mediated rescue of smc6-74: pathways required for base alkylation repair

Post-replication repair pathways enable tolerance of alkylated bases, that is,

bypass but not repair of the actual lesion. In S. pombe, there is ample genetic evidence

that alkylated bases are repaired by recombinational mechanisms (for examples, see

JANG et al. 1994; KAI and WANG 2003; MORIKAWA et al. 2004; SMEETS et al. 2003;

TSUTSUI et al. 2000). As stated above, we were not able to assay mutants in the

homologous recombination pathway due to epistasis with smc6-74. However, two

complexes function redundantly in the recombination pathway that aid in the loading of

Rhp51 into the nucleoprotein filament on single-stranded DNA. One complex consists of

Swi5 and Srf1, the other consists of Rhp55 and Rhp57 (homologs of S. cerevisiae

RAD55 and RAD57), and only when both complexes are absent do cells phenocopy

rhp51∆ (AKAMATSU et al. 2003). As shown in Fig 5, suppression of smc6-74 by Brc1

clearly required Rhp57, but not Swi5. Interestingly given the epistasis between smc6-74

and rhp51∆ or rhp54∆, both the rhp57∆ smc6-74 and swi5-39 smc6-74 double mutants

were significantly more MMS sensitive than either parent. These data are consistent with

the model that Brc1 promotes recombinational repair of alkylated bases in smc6-74

mutants in G2 phase, following bypass of the lesion during S-phase by the post-

replication repair pathway. It is also possible that the recombination pathway is

processing stalled replication forks in S-phase and promoting replication restart.

Stability of stalled replication forks, and the successful resumption of DNA

replication are reliant on two redundant groups of response enzymes. The predominant

response is via the RecQ-type helicases, encoded by rqh1 in S. pombe, together with

Topoisomerase III (KHAKHAR et al. 2003). rqh1 is not an essential gene, and MMS

sensitivity assays showed suppression of an rqh1∆ smc6-74 by Brc1 back to the

sensitivity seen for the rqh1∆ single mutant (data not shown). This is in keeping with our

observed lack of epistasis between brc1∆ and rqh1∆ (Fig 2D). Three dimeric protein

complexes have been identified by genetic studies in S. cerevisiae and S. pombe that

act redundantly with Sgs1 and Rqh1 respectively (COULON et al. 2004; DOE et al. 2002;

MULLEN et al. 2001). These complexes include two structure-specific nucleases,

Mus81/Eme1 and Slx1/Slx4, which have a preference to cleave structures resembling

stalled replication forks, and a third complex, Slx5/Slx8, of unknown function. We deleted

the S. pombe slx8 gene, which resulted in extremely slow growing and poorly viable

cells (data not shown), and so no further genetic analyses were attempted. However, we

could assay the requirement for the other complexes using mus81∆ and slx1∆ strains.

For slx1∆ smc6-74, we observed very little suppression of by Brc1 in both chronic and

acute exposure to MMS (Fig 6A, C), a somewhat surprising result given that slx1∆ by

itself shows wildtype levels of MMS sensitivity (COULON et al. 2004). For mus81∆ smc6-

74, nearly complete suppression was observed (Fig6B, C), though compared to the

smc6-74 parent, we reproducibly observed an increased MMS sensitivity in the double

mutant overexpressing Brc1 in several independent experiments, assayed by both

chronic and acute MMS exposure. These data show a strong requirement for Slx1, and

a more modest requirement for Mus81, in order for Brc1 overexpression to suppress

smc6-74.

In the course of these experiments we noted that smc6-74 slx1∆ double mutants

were substantially more MMS sensitive than either smc6-74 alone, or smc6-X slx1∆

double mutants which were identical to smc6-X single mutants (Fig 7). Again, this is

particularly notable as slx1∆ cells are not hypersensitive to MMS (COULON et al. 2004),

and smc6-X is not suppressible by Brc1 overexpression (VERKADE et al. 1999).

Combined, these data suggest structures that are efficiently processed by

Slx1/Slx4 are prevalent in smc6-74 cells grown in the presence of MMS, and that Brc1

may promote their processing via Slx1/Slx4 into the Rhp57-dependent arm of

recombination.

Brc1-mediated suppression of the UV-C hypersensitivity of smc6-74

The repair of alkylation damage, and its relationship to replication fork stalling,

largely investigates DNA repair and tolerance mechanisms during DNA replication.

However, Brc1 overexpression can also suppress the UV-C hypersensitivity of smc6-74.

In these experiments, an asynchronous culture is irradiated, but in S. pombe this

represents approximately 75% of cells in G2 phase, and only 10% in S-phase. Thus, the

Brc1-mediated suppression of repair defects in smc6-74 is not confined to S-phase

damage, even though brc1∆ cells are not hypersensitive to UV-C in G2. We therefore

took a similar genetic approach to the above experiments using MMS to investigate

which genes are required to suppress defective UV-C lesion processing in smc6-74

cells, bearing in mind that the majority of these responses are occurring in G2. The data

are summarized in Table 1, with example survival curves in Fig 8.

As seen with MMS exposure, the suppression of smc6-74 by Brc1

overexpression was wholly dependent on rhp18, though independent of ubc13. In the

case of MMS, similar observation predicted a role for translesion synthesis. However,

these polymerases are largely not required in G2, and hence their absence does not

result in a significant UV-C hypersensitivity in asynchronous cultures. Not surprisingly

therefore, Brc1 overexpression still largely suppressed the UV-C hypersensitivity of

smc6-74 in the absence of polymerases η, ζ, and κ (3TLS∆), with the residual sensitivity

presumably accounted for by the G1 and S-phase cells in the culture. Moreover, we

consistently observed that Brc1 overexpression increased the sensitivity of the 3TLS∆

strain to UV-C. Brc1-mediated channeling during G1 and S-phase of the lesions into a

pathway that is dependent on these polymerases, which becomes a toxic event in their

absence, may explain this. Further, unlike the case for alkylation damage, we saw no

effect of deleting rad9. The checkpoint response itself was clearly not required, as we

observed complete suppression of chk1∆ smc6-74.

We next assayed the Rhp57- and Swi5-dependent recombination pathways.

Epistasis again excluded assays with downstream genes in recombination pathway such

as rhp51. As with alkylation damage, Brc1-mediated suppression of smc6-74 UV-C

hypersensitivity was dependent on rhp57, but in this case swi5-39 smc6-74 mutants

were partially defective in the suppression assays. Further, slx1∆ smc6-74 cells showed

little suppression, despite the observation that slx1∆ cells show wildtype sensitivity to

UV-C, and mus81∆ smc6-74 cells showed considerable though incomplete suppression.

In the UV-C irradiated S-phase cells (~10% of the population), the recombination genes

may also process stalled replication forks and ensure restart of replication.

Together these data suggest that UV-C irradiated smc6-74 cells accumulate

aberrant DNA structures that can be resolved by recombination, and that such a

recombination pathway is promoted by the concerted action of Brc1, Slx1, Rhp18 and to

a lesser extent, Mus81. As neither brc1∆ nor slx1∆ cells are hypersensitive to UV-C, the

structures forming in the background of smc6-74 must either not form in wildtype cells,

or are rapidly processed by another pathway, perhaps requiring wildtype smc6.

Discussion

The Smc5/6 complex, although relatively poorly understood at this stage, is as

fundamental to chromosome dynamics as its relatives condensin and cohesin. The

genetic relationships between smc6 and brc1 in S. pombe described here provide a

framework on which future molecular studies can elucidate molecular details to the

precise function of the Smc5/6 complex. Previous studies have implicated a role for the

Smc5/6 complex in recombinational repair, though how it could influence the core

recombination machine is unknown. By studying how overexpression of Brc1 can

suppress the hypersensitivity of the checkpoint and repair defective smc6 allele smc6-

74, we can build a model of the defect in smc6-74 cells, and thus dissect the function for

the wildtype Smc5/6 complex.

First, considering the response to the alkylating agent MMS, cells need to

contend with both the physical interference with the replication complexes, and the

mechanisms by which the lesions are repaired. There is considerable evidence in S.

pombe that whilst translesion synthesis polymerases can bypass alkylated bases, based

on the sensitivity of various DNA repair mutants, their repair is largely dependent on

recombination and, to a lesser extent, nucleotide excision repair (NER). In order for Brc1

to suppress the MMS hypersensitivity of smc6-74, translesion synthesis polymerases

are required, suggesting that the actual repair occurs in the subsequent G2 phase

following lesion bypass during S-phase. The requirement for Rhp57 for the suppression,

and the published observations that this protein, in complex with Rhp55 aids in the

loading of Rhp51 in G2 (AKAMATSU et al. 2003), is consistent with a repair in G2

following bypass of the initial lesions. However, there is a strong requirement for Slx1 for

this suppression, and for MMS resistance in smc6-74 but not smc6-X. Slx1, which with

its partner Slx4 cleaves Y-shaped structures characteristic of replication forks and

recombination intermediates (COULON et al. 2004). Upon Brc1 overexpression, Slx1/Slx4

may cleave such accumulating structures in smc6-74 cells to recruit the recombination

machinery, or to process an intermediate of recombination. It is tempting to speculate

that in wildtype cells, the Smc5/6 complex normally functions in such recruitment of

recombination factors to stalled replication forks or alkylated lesions. Notably, smc6-74

substantially increased the MMS sensitivity of rhp57∆ and swi5-39 strains, which indeed

are defective in initiating recombination, suggesting that wildtype Smc6 function may be

critical for recombination in these backgrounds. Mus81, which with its partner Eme1 also

cleaves Y-shaped structures albeit with a different specificity (GAILLARD et al. 2003;

KALIRAMAN et al. 2001; OSMAN et al. 2003), played a minor role in the Brc1-mediated

suppression of smc6-74. Thus, Mus81/Eme1 may be essential for the processing of a

minority of lesions in smc6-74, perhaps explaining our observed epistasis between

mus81∆ and brc1∆. Mus81/Eme1 is essential for the viability in the hypomorphic nse1-1,

nse2-1, nse3-1 and nse4-1 (rad62-1) mutants (MORIKAWA et al. 2004; PEBERNARD et al.

2004). We have not however observed this for smc6-74 or smc6-X, and so the

differential requirement for a particular structure specific nuclease likely reflects the

accumulation of different lesions depending on the nature of the mutation in a particular

Smc or non-Smc subunit.

In the case of UV-C irradiation in G2 cells, the requirements for suppression were

similar to that for MMS with two exceptions. First, the translesion synthesis polymerases

were not required, which is not surprising since they are thought to function primarily

during S-phase. Second, Swi5 was required for complete rescue, which it was not for

MMS. Regardless, the observations for UV-C sensitivity also suggest that Slx1/Slx4

processes a structure in smc6-74 cells that promotes recombination repair. We assayed

UV-C sensitivity in G2 and S-phase brc1∆ cells, but were unable to demonstrate any

sensitivity. This suggests that Brc1 is only required to repair UV-C induced lesions once

inappropriately processed into a structure in smc6-74 cells that perhaps resembles those

present in MMS treated cells, and that this uses Slx1-initiated recombination. Notably,

we found no evidence to link Smc5/6 or Brc1 to the NER pathway.

The absolute dependence on Rhp18 is in keeping with or previous findings in

synchronous populations of cells that this protein is required for UV-C tolerance

throughout the cell cycle (VERKADE et al. 2001). In the case of MMS treatment, the lack

of requirement for ubc13 can easily be rationalized on the channeling of lesions into the

tolerance pathway controlled by translesion synthesis polymerases. Importantly

however, the lack of requirement for both ubc13 and translesion synthesis for the UV-C

response suggests a hitherto unseen pathway acting downstream of Rhp18 during G2

that awaits identification.

In wildtype cells, rqh1 plays key roles in rescuing stalled replication forks in S-

phase, and in the repair of UV-C lesions in G2 (KHAKHAR et al. 2003; MURRAY et al.

1997). As the Brc1-mediated rescue of smc6-74 does not require rqh1, the defect in

smc6-74 is not resolved using this pathway. It is therefore possible that wildtype Smc5/6

may contribute to Rqh1 function. Curiously, whilst rqh1∆ slx1∆ show a synthetic lethality

(COULON et al. 2004), rqh1∆ brc1∆ cells are viable though show a high degree of mitotic

abnormalities and grow significantly slower than wildtype cells. Thus, whilst Brc1 can

stimulate Slx1-dependent repair in smc6-74, it is not absolutely required for Slx1 activity

in rqh1∆ cells.

With its many BRCT domains, Brc1 is predicted to bind to one or more

phosphoproteins (MANKE et al. 2003). We have assayed for physical interaction between

Brc1 and Slx1 and failed to detect, even with overexpression. We have also attempted to

assay interaction with Slx4, but have found that epitope tagging of Slx4 results in a non-

functional and extremely unstable protein.

In summary, from these observations we propose a model (Fig 9) that in wildtype

cells, the Smc5/6 complex functions in the recruitment of recombination complexes to

lesions. Brc1 controls an alternative pathway to initiate recombination utilizing primarily

Slx1, and a yet to be deciphered sub-branch of the post-replication repair pathway.

Clearly, it will be important to dissect molecular details to explain the genetic

observations described here.

Acknowledgments

We are particularly grateful to Drs A.M. Carr and H. Shinagawa for the provision of

strains and discussions. M.O’C is a Scholar of the Leukemia and Lymphoma Society.

K.M.L. is supported by an NIH/NCI training grant T32 CA78207. This work was

supported by grants from the Australian Research Council (A00103857), the Peter J.

Sharp Foundation for Melanoma Research and from Cancer Research UK

(SP2396/0101).

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Legends to the figures

Figure 1. Brc1 is required for resistance to radiomimetic drugs. Drug sensitivity assays

were performed on YES medium using wildtype and chk1∆ controls. Plates were

photographed after 4 days at 30°C. Micrographs show brc1∆ cells after 6 hours of drug

treatment in liquid culture. Bar = 10µm.

Figure 2. Epistasis of the MMS sensitivity of brc1∆. Acute (liquid) and chronic (plate)

assays of MMS sensitivity brc1∆ combined with: A. rhp51∆; B. mus81∆; C. rhp18∆; and

D. rqh1∆. A control (0% MMS), and a representative plate from an MMS dilution series

are shown. A. brc1∆ rhp51∆ shows similar sensitivity as the rhp51∆ parent. B. Similar

epistasis is seen with mus81∆. C. rhp18∆ and brc1∆ synergize for MMS sensitivity, but

the double mutant grows as wildtype cells on the control plate. D. rqh1∆ and brc1∆

synergize for MMS sensitivity, but the double mutant is growth retarded on the control

plate.

Figure 3. Brc1-mediated suppression of smc6-74 is dependent on Rhp18, but

independent of Ubc13. The indicated strains transformed with vector or pBrc1 were

assayed at a range of MMS doses. Shown are control (0% MMS) and 0.0025% MMS.

Plates were photographed after 4 days at 30°C.

Figure 4. Translesion synthesis polymerases are required for Brc1-mediated

suppression of the MMS hypersensitivity of smc6-74. In each case a range of MMS

concentrations were assayed, and a control (0% MMS) and a representative MMS

concentration is shown. Plates were photographed after 4 days at 30°C. A. Strains

lacking polymerases η, ζ, and κ (encoded by eso1, rev3 and dinB) or dinB alone were

crossed to smc6-74 and assayed for Brc1-mediated suppression. Eso1∆ and rev3 single

mutants showed no defect in suppression, and eso1 dinB and rev3 dinB double mutants

behaved identically to dinB single mutants (not shown). B. rad9 and rad17 are required

for Brc1-mediated suppression of smc6-74, but chk1 is not (C).

Figure 5. The Rhp57 recombination pathway is required for Brc1-mediated suppression

of the MMS hypersensitivity of smc6-74. The indicated strains were tested for MMS

sensitivity for 4 days at 30°C. Note no suppression and enhanced sensitivity of the

smc6-74 rhp57∆ double (top panel), but complete suppression of the enhanced

sensitivity of the swi5-39 smc6-74 double mutant (bottom panel).

Figure 6. Structure-specific nucleases are required for Brc1-mediated suppression of

the MMS hypersensitivity of smc6-74. Brc1 suppression assays using the indicated

strains were performed for both chronic (A, B) and acute (C) exposure to MMS. For

chronic exposure, a range of MMS concentrations were assayed, and representative

plates are shown. slx1∆ almost completely abolished suppression by both assays.

mus81∆ results in a incomplete suppression.

Figure 7. Slx1 is required for MMS resistance of smc6-74 but not smc6-X. MMS

sensitivity of the indicated strains was assayed over a range of MMS concentrations at

30°C for 4 days.

Figure 8. Requirements for the Brc1-mediated suppression of the UV-C hypersensitivity

of smc6-74. Survival data, normalized to unirradiated controls, is shown for the indicated

strains (n=3-6). Effective suppression moves the open triangles to the survival of open

circles.

Figure 9. Model for Brc1-mediated DNA damage responses. DNA alkylation damage in

S-phase leads to repair through either Smc5/6 or Brc1 dependent pathways, both of

which involve homologous recombination (HR). In the Brc1 dependent pathway Mus81

is likely to act at a stalled replication fork and then replication restarts following strand

invasion by the HR machinery but it may be required to resolve recombination

intermediates. In the smc6-74 mutant, recombination repair of alkylated bases is

compromised and wildtype levels of Brc1 are unable to fully compensate, leading to cell

death. Overexpression of Brc1 (OP-Brc1) suppresses the defect in smc6-74 cells by

either more efficiently channeling repair of the primary lesion through the Brc1

dependent pathway or by channeling the intermediates generated in the defective

Smc5/6 pathway into alternative repair pathway(s). This requires Rhp18 (but not Ubc13),

TLS, and Rhp57-mediated HR, and the structure specific nucleases Slx1/4 and to a

lesser extent Mus81/Eme1. It is envisaged that lesions are either processed by Slx1 or

Mus81 or bypassed by TLS (Rhp18-dependent) and subsequently repaired in G2 by HR.

Lethal recombination intermediates generated in smc6-74 could be processed by Slx1/4

(and possibly Mus81) and Rhp57-dependent HR. These intermediates are specific to the

smc6-74 mutant and require Slx1 for processing. Repair of UV lesions in G2 by HR

requires the Smc5/6 complex, but not Brc1, as brc1∆ cells are not hypersensitive to UV-

C. In the smc6-74 mutant, UV irradiation leads to cell death and this is suppressed by

overexpression of Brc1. In this case suppression requires Rhp18 (but not Ubc13 or

TLS), Rhp57-mediated (and to a lesser extent Swi5-mediated) HR, and the structure

specific nucleases Slx1 and Mus81. As above overexpression of Brc1 could channel

repair into an alternative pathway and also channel the lethal intermediates generated in

the smc6-74 mutant into a recombination-based pathway.

Table 1. Brc1 suppression of smc6-74. Summary of suppression of smc6-74 by Brc1 overexpression. MMS sensitivity was determined over a range of concentrations appropriate for parental strains. UV-C sensitivity was determines from 0-250 J/m2. Degree of rescue was estimated from survival curves. +++++ = rescue to sensitivity of allele tested (>95% suppression); - = same as vector only control (0% suppression) ; +/- = marginal (<5%) suppression; + = 5-20%, ++ = 20-40%, +++ = 40-80%, and ++++ = 80-95% suppression. ND: Not determined. Allele tested MMS UV chk1∆ +++++ +++++ cds1∆ +++++ +++++ rad13-A +++++ +++++ rqh1∆ +++++ +++++ rhp18∆ - - slx1∆ +/- +/- mus81∆ +++ +++ ubc13∆ +++++ +++++ TLS 3∆ + ++++ dinB∆ (pol κ) +++ +++++ rad3∆ + +++++ rad9∆ + +++++ rad17∆ + ND rhp57∆ - - swi5-39 +++++ ++ rhp51∆ Epistatic Epistatic rhp54∆ Epistatic Epistatic rad2-44 Epistatic Epistatic rad32∆ +++++ +++++

wildtypechk1∆brc1∆smc6-74smc6-74 + pBrc1

Control 10µM CPT5mM HU30ng/ml 4-NQO0.01% MMS

brc1

∆

0 20 40 60Minutes in 0.05% MMS

100

10

1

% S

urvi

val

0 20 40 60Minutes in 0.05% MMS

100

10

1

0.1

% S

urvi

val

0 20 40 60Minutes in 0.05% MMS

100

10

1

0.1

% S

urvi

val

0 20 40 60Minutes in 0.05% MMS

100

10

1

0.1

0.01

0.001

% S

urvi

val

0.00075% MMS

0.001% MMS 0.001% MMS

0.00075% MMS

Control Control

ControlControl

Key:Plates (top to bottom): Graph symbol:Wildtype c brc1∆ n gene X brc1∆ geneX ▲

c

A. rhp51∆

D. rqh1∆

B. mus81∆

C. rhp18∆

ubc13∆, vectorubc13∆, pBrc1smc6-74, vectorsmc6-74, pBrc16-74;13∆, vector6-74;13∆, pBrc1

Control 0.0025% MMS

Control 0.0025% MMS

rhp18∆, vectorrhp18∆, pBrc1smc6-74, vectorsmc6-74, pBrc16-74;18∆, vector6-74;18∆, pBrc1

Control 0.0025% MMSsingle, vector

single, pBrc1

double, vector

double, pBrc1

Controls*

chk1

∆

Control 0.0015% MMSsingle, vector

single, pBrc1

double, vector

double, pBrc1

Controls*

single, vector

single, pBrc1

double, vector

double, pBrc1

Controls*

rad9

∆ra

d17∆

Control 0.0075% MMSsingle, vector

single, pBrc1

double, vector

double, pBrc1

Controls*

single, vector

single, pBrc1

double, vector

double, pBrc1

Controls*

3TLS

∆di

nB∆

*Controls are the third dilution of: wildtype, vector; wildtype, pBrc1; smc6-74, vector; smc6-74, pBrc1

smc6-74, vectorsmc6-74, pBrc1rhp57∆, vectorrhp57∆, pBrc1smc6-74; rhp57∆, vectorsmc6-74; rhp57∆, pBrc1

wildtype, vectorwildtype, pBrc1swi5-39, vectorswi5-39, pBrc1smc6-74; swi5-39, vectorsmc6-74; swi5-39, pBrc1

Control 0.001% MMS0.00075% MMS0.0005% MMS

100

10

1

0.10 20 40 60

Minutes in 0.05% MMS

% S

urvi

val

slx1∆

wildtypeslx1∆ or mus81∆smc6-74slx1∆ or mus81∆;smc6-74

vecto

r

pBrc1

% S

urvi

val

0 20 40 60Minutes in 0.05% MMS

100

10

1

0.1

0.01mus81∆

C.

Control 0.0015% MMSA. slx1∆

single, vector

single, pBrc1

double, vector

double, pBrc1

Controls*

ControlB. mus81∆

0.001% MMSsingle, vector

single, pBrc1

double, vector

double, pBrc1

Controls**Controls are the third dilution of: wildtype, vector; wildtype, pBrc1; smc6-74, vector; smc6-74, pBrc1

0.002% MMS

0.0005% MMS

wildtypesmc6-74smc6-Xslx1∆smc6-74 slx1∆smc6-X slx1∆

Control

0.0005% MMS 0.00075% MMS

0.001% MMS 0.0015% MMS

A.

100

10

1

0.1

0.01

100

10

1

0.1

0.01

0.001

% s

urvi

val

0 50 100 150 200 250

rhp18∆ ubc13∆

UV-C (J/m2)0 50 100 150 200 250

UV-C (J/m2)

0 50 100 150 200 250UV-C (J/m2)

100

10

1

0.1

0.01 chk1∆

100

10

1

0.10 50 100 150 200 250

UV-C (J/m2)

% s

urvi

val

slx1∆

100

10

1

0.10 50 100 150 200 250

UV-C (J/m2)

mus81∆

wildtypesinglesmc6-74double

vecto

r

pBrc1

% s

urvi

val

100

10

1

0.1

0.01

0.001rhp57∆

100

10

1

0.1

0.01swi5-39

0 50 100 150 200 250UV-C (J/m2)

0 50 100 150 200 250UV-C (J/m2)

0 50 100 150 200 250UV-C (J/m2)

100

10

1

0.13TLS∆

% s

urvi

val

100

10

1

0.1

0.01

0.001rad9∆

0 50 100 150 200 250UV-C (J/m2)