Role of the error-free damage bypass postreplication repair pathway in the maintenance of genomic stability

Mutation Research 532 (2003) 117–135

Role of the error-free damage bypass postreplication repair pathway in the maintenance of genomic stability Marina Smirnova, Hannah L. Klein∗ Department of Biochemistry, Kaplan Comprehensive Cancer Center, New York University School of Medicine, 550 First Avenue, New York, NY 10016, USA Received 29 June 2003; received in revised form 8 August 2003; accepted 12 August 2003

Abstract The postreplication repair pathway (PRR) is composed of error-free and error-prone sub-pathways that allow bypass of DNA damage-induced replication-blocking lesions. The error-free sub-pathway is also used for bypass of spontaneous DNA damage and functions in cooperation with recombination pathways. In diploid yeast cells, error-free PRR is needed to prevent genomic instability, which is manifest as loss of heterozygosity (LOH) events of increased chromosome loss and recombination. Homologous recombination acts synergistically with the error-free damage avoidance branch of PRR to prevent chromosome loss. The DNA damage checkpoint gene MEC1 acts synergistically with the PRR pathway in maintaining genomic stability. Integration of the PRR pathway with other cellular pathways for preventing genomic instability is discussed. In diploid strains, the most dramatic increase is in the abnormality of chromosome loss when a repair or damage detection pathway is defective. © 2003 Elsevier B.V. All rights reserved. Keywords: Saccharomyces cerevisiae; Damage avoidance; Postreplication repair; Genomic stability

1. Introduction The postreplication repair pathway (PRR) is one of several processes used to tolerate DNA damage. The pathway derives its name from observations regarding cellular responses to DNA damaging agents that result in lesions causing the replication machinery to stall. The unrepaired DNA lesions give rise to single-strand gaps, which are subsequently filled in using the PRR pathway. Thus, the PRR pathway is characterized by the ability to convert low molecular weight single-stranded DNA into high molec∗ Corresponding author. Tel.: +1-212-263-5778; fax: +1-212-263-8166. E-mail address: [email protected] (H.L. Klein).

ular weight species [1,2]. Although the lesions of single-stranded gaps are repaired, the damage still remains in the template strand, so the PRR pathway is a lesion bypass pathway, or a damage avoidance pathway, not a true repair pathway in the sense that the initial lesion is removed. Mutants in the PRR pathway are sensitive to variety of DNA damaging agents, including ionizing radiation, UV irradiation, alkylating agents such as methylmethane sulfonate (MMS), and DNA cross-linking agents [3]. The PRR pathway of Saccharomyces cerevisiae is often called the RAD6 pathway, named after the RAD6 gene identified within this epistasis group of ionizing radiation sensitivity [4,5]. The general function of the PRR pathway in S. cerevisiae has recently been reviewed [6]. A brief summary of the pathway is presented

0027-5107/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2003.08.026

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here, with emphasis on the error-free damage avoidance branch. A review of this pathway with emphasis on bypass replication at stalled replication forks can be found in this issue [7]. The RAD6 pathway is conserved throughout evolution [6]. In Escherichia coli, postreplication repair uses the SOS response and bypasses replication-blocking DNA lesions through translesion DNA synthesis using specialized DNA polymerases and RecA-dependent replication restart [8]. In eukaryotes, the genes defined in the yeast S. cerevisiae have counterparts in Schizosaccharomyces pombe [9] and mouse [10] that appear to act in similar DNA damage avoidance pathways. The PRR pathway is controlled by the RAD6 and RAD18 genes, whose products form a heterodimer with DNA binding activity and ubiquitin-conjugating activity [11,12]. The binding of the Rad6/Rad18 complex to single-stranded DNA-containing a lesion is thought to initiate use of the PRR pathway by ubiquitination of target proteins that act downstream. The error-free sub-pathways are composed of a translesion synthesis (TLS) DNA polymerase encoded by the RAD30 gene, and the damage avoidance sub-pathway which requires function of the RAD5, MMS2, UBC13 and SRS2 genes as well as DNA polymerase ␦, PCNA and homologous recombination (HR) functions [6,13–18]. Interestingly, the MMS2 and UBC13 genes are also related to ubiquitin-conjugating enzymes [13,19,20] and a Mms2/Ubc13 complex may modify Rad6 or other factors in the PRR pathway [13–15]. The Rad5 protein is a member of the SWI/SNF superfamily and has ATPase activity and a RING finger motif [21,22]. The precise function of Rad5 in the PRR pathway has not been fully defined. It may facilitate chromatin remodeling to help repair proteins bind to DNA repair substrates. It has been proposed to function in template switching during stalled DNA replication [23]. Rad5 has also been linked to protein modification. PCNA has been shown to be a target for modification by many protein products of the PRR pathway. PCNA is mono-ubiquitinated by Rad6 and Rad18, multi-ubiquitinated by Rad6, Rad18, Mms2, Ubc13 and Rad5, and sumoylated by Ubc9 [24]. The multi-ubiquitination of PCNA is necessary for RAD6-dependent DNA repair via the error-free sub-pathway that involves MMS2, UB13 and RAD5.

1.1. Overlap between PRR and HR in damage avoidance The E. coli SOS DNA damage response pathway uses homologous recombination to tolerate DNA damage, by recombination-mediated replication fork restart or template switching [8,25–32]. The yeast rad6 and rad18 mutants show increased spontaneous mitotic recombination for a multiplicity of substrates [33–37], suggesting that the PRR damage avoidance pathway is an alternative mode of damage bypass to homologous recombination. PRR damage bypass may involve template switching [23] and the RAD5 gene is proposed to be a key factor in this damage avoidance replication [23,38]. rad5 mutants generally have not been reported to have any recombination phenotype using unique gene substrates, although Kupiec and co-workers found that a rad5∆ mutant had a five-fold increase in direct repeat recombination [35]. Indeed, one of the commonly used yeast strains, W303, harbors a mutation in the RAD5 gene, which is slightly hypomorphic [39], and this allele had no effect on gene conversion or crossing over, compared to an isogenic RAD5 strain (HLK, unpublished observations). While a rad5∆ mutant has no observable effect on allelic recombination (HLK, unpublished observations), a rad5 mutant has been reported to show increased slippage in a simple repeat sequence [21]. This could be a consequence of slipped template switching in the rad5 mutant. The direct repeat recombination events studied by Kupiec and co-workers could result from several events, which include replication slippage and unequal sister chromatid exchange [35]. Thus, their findings may be similar to those reported by Johnson et al. for simple repeat recombination in rad5 mutants [21]. The same study also found that rad18∆ mutants were increased for direct repeat recombination [35], and that rad5∆ and rad18∆ mutants were increased for spontaneous gene conversion rates, although gene conversion was measured between Ty elements and the increase in gene conversion between dispersed Ty elements could be a special feature of Ty sequences. Ectopic gene conversion between non-Ty sequences was increased in these same mutants, although only about three-fold [35]. All of the gene conversion events were dependent on the homologous recombination genes RAD51 and RAD52, while the direct repeat events were only dependent on RAD52 [35].

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These findings suggest that the direct repeat events observed in rad5∆ and rad18∆ mutants were the result of a single-strand annealing event or replication slippage, but not a HR event requiring strand invasion. The fact that the presence of Rad5 and Rad18 proteins suppresses gene conversion suggests that either more lesions are formed when these proteins are missing, or that the PRR pathway actively suppresses recombination by repairing spontaneous lesions in a way that does not result in a genetically distinguishable recombination product. Another study looked at recombination in a rad18∆ mutant and concluded that a rad18 diploid showed increased interchromosomal gene conversion and crossing over, but that intrachromosomal recombination was unaffected [37]. It is not clear why the Kupiec and co-workers observed increased direct repeat recombination in the rad18∆ haploid mutant [35], while the Wintersberger and co-workers did not [37]. However, it should be noted that recombination in the rDNA locus was increased in a rad18 mutant strain [37]. Since the rDNA has a large number of repeats, the target size for repeat sequence recombination is great. Since these two groups used different repeat sequence reporters for direct repeat recombination, the difference in rate increase in the two studies might reflect the size of the duplicated sequence or the selection for recombinant segregants. 1.2. Role of SRS2 in regulating repair by the PRR pathway The SRS2 gene was first identified in a screen for suppressors of the damage sensitivity of a rad6 mutant [40]. It was subsequently re-isolated in a screen for suppression of the damage sensitivity of rad18 mutants [41] or hyper-recombinant mutants [42]. Genetic studies showed that SRS2 functions in the error-free branch of the PRR pathway to regulate repair of a lesion through gap repair rather than allowing the substrate to be channeled to a HR repair pathway. Thus, srs2 mutants could suppress the damage sensitivities of rad5, rad6, and rad18 mutants of the PRR pathway [40–42]. Similar to the rad6 and rad18 mutants, srs2 mutants are increased in intrachromosomal and interchromosomal gene conversion [42]. Cloning of the SRS2 gene revealed that the Srs2 protein had homology to the bacterial DNA helicase UvrD [41,43],

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and in vitro activity of the purified protein showed it to be a 3 –5 DNA helicase [44]. The suppression phenotype of srs2 mutants is dependent on functional HR, giving further support to the channeling hypothesis [41,42,45,46]. Suppressors of the srs2 mutant channeling function were found to cluster in the RAD51 gene [47,48], which encodes a strand exchange activity essential for most HR. These findings have lead to the hypothesis that the Srs2 DNA helicase has antirecombinase activity by antagonizing Rad51 protein or some heteroduplex intermediate in homologous recombination. Recently, in vitro experiments studying the action of Srs2 protein on Rad51 protein assembled as a filament on single-stranded DNA have shown that Srs2 protein can disrupt Rad51 filaments [49,50]. Although it would seem that SRS2 regulates use of the error-free PRR pathway by preventing recombination, not all PRR mutants fit nicely into this scenario. As discussed earlier, suppression of rad6 and rad18 DNA damage sensitivities occurs in srs2 mutants and this suppression requires active HR functions [41,43,46]. However, the hyper-recombination phenotype of rad5 and rad18 mutants requires SRS2 function, whether or not the hyper-recombination is dependent on RAD51 [35]. In genetic terms, the srs2∆ recombination phenotype was epistatic to that of rad5∆ and rad18∆ mutants. This suggests that SRS2 has a role in error-free PRR that is more extensive than regulating repair of a gapped substrate, or that recombination events seen in a rad51 rad5 mutant, for example, are not identical to those seen in a RAD51 rad5 (or RAD51 rad18) mutant. It is possible that Rad51 protein itself is inhibitory to the single-strand annealing type of direct repeat recombination event that is elevated in the rad5 (or rad18) mutant (Fig. 1). This might be the postulated pro-recombination activity of SRS2. Thus, hyper-recombination will be lost in srs2 rad5 and srs2 rad18 mutants, even though repair events may be channeled to the HR pathway. SRS2 also functions in regulating use of the error-prone part of the PRR pathway. The increased spontaneous mutation rate of rad5 and rad18 mutants is dependent on the error-prone DNA polymerase ␨, encoded by the REV3 and REV7 genes [35]. The hypermutability of these strains requires SRS2, but does not require the HR gene RAD51 [35]. This is expected, according to the channeling model of Fig. 2. Detailed epistasis studies have shown that SRS2 is required

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End processing of a DSB to generate 3' single-stranded tails

Single-stranded DNA coated with Rad51 protein

Removal of Rad51 filament and annealing of single strands

Trimming of 3' heterologous tails and ligation to generate a deletion

Fig. 1. Model for SSA recombination. Thick gray lines depict the repeated sequences. After a double-strand break is introduced, the ends are resected to give 3 protruding tails. These can be coated with Rad51 protein for repair by homologous recombination. If no homologous sequence is present for strand invasion, then complementary single-strand regions of the repeated sequence can reanneal, after Rad51 protein is removed. The Srs2 protein is proposed to be involved in Rad51 removal. After the complementary sequences have annealed, single-strand tails are removed by structure-specific nucleases and the nicks are sealed by DNA ligase.

ssDNA gapped substrate

Srs2 present Srs2

antagonizes Rad51 binding

Srs2 absent

Rad51 binds ssDNA

Rad18 Rad6

processing to recombination intermediate error-free bypass error-prone bypass Damage bypass without recombination

Damage repair by recombination

Fig. 2. Model for regulation of PRR pathway by Srs2 protein. When Srs2 is present, it prevents Rad51 from forming a filament on the ssDNA present in a gap, and thus allows Rad6 and Rad18 to bind and promote error-free or error-prone lesion bypass. When Srs2 is absent, Rad51 is not prevented from binding and stimulating homologous recombination. It is not known if the ssDNA gap is processed to a double-strand break prior to Rad51 assembly.

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for the error-free portion of the PRR pathway and for RAD6 and RAD18 activation of the PRR pathway [14,16,34,43,46]. However, SRS2 is not required for the error-prone damage-induced mutagenesis branch of the PRR pathway [14,16], suggesting that Srs2 protein only targets substrates for Rad6/Rad18 binding that can be repaired by the error-free sub-pathway of PRR. In the absence of error-free repair, Srs2 may appear to promote error-prone translesion synthesis by inhibiting Rad51-promoted HR, but the promotion of error-prone repair by Srs2 may be a secondary consequence of the failure of the error-free repair pathway. Pol32, a subunit of DNA polymerase ␦, has been shown to be involved in the error-prone repair sub-pathway of PRR [51]. Interestingly, Srs2 has been shown to interact with Pol32, a subunit of DNA polymerase ␦ [51], and this interaction may be important for determining whether error-free or error-prone repair is used for PRR. 1.3. Regulation of repair by mating type Diploid yeast cells are more resistant to DNA damage than haploid cells. This is the result of a homolog chromosome that is available as a repair template through HR during the G1 phase of the cell cycle. Consequently, the increased diploid damage resistance is dependent on the genotype at the mating type locus MAT, which increases HR when heterozygous a/␣ [52]. Since HR is an alternative to PRR for repair of spontaneous damage, regulated by SRS2, mating type and haploid/diploid effects come in to play in the damage sensitivity of PRR mutants. rad18 diploid mutants are more sensitive to ionizing radiation when expressing only one mating type, compared to isogenic rad18 diploids that express both mating types [52]. This presumably is due to increased HR. One caveat here is that we do not know what type of recombination event is occurring to repair the induced lethal damage in the rad18 mutants. When gene conversion is measured in rad18 mutants, the increased rate observed is not dependent on heterozygosity at MAT [37]. The type or amount of damage may influence the efficiency of repair by HR. Whatever the explanation, the use of HR as a back-up repair pathway for PRR is not always regulated by mating type. Rescue by HR may not always involve recombination repair with the homolog chromosome. Radiation

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sensitivity of a haploid MATa rad6∆ mutant could be suppressed by expressing a plasmid-based copy of MATα. This suppression required an active HR system [53]. Another important factor to consider is that not all HR events in PRR mutants may be beneficial for survival. In support of this viewpoint is the finding that the UV damage sensitivity of srs2∆ mutants is higher in diploids expressing both mating types [52]. These diploids should have increased HR, and in the context of a srs2∆ diploid, some HR events must be deleterious. In the absence of a functional PRR pathway, increased HR can suppress damage sensitivity, but for the SRS2 regulator, increased HR is harmful. Some induced damage may be repaired in G1 by HR, or in G2 in haploid strains by HR, but damage that stalls or blocks replication must be repaired in a way that not only removes the stalling lesion, but also restores the replication fork. Exchange HR, especially HR between homolog chromosomes, may repair the lesion but destroy the fork. 1.4. Additional pathway for damage avoidance Double mutant yeast strains with mutations in HR and PRR genes are viable, suggesting that other pathways can repair spontaneous damage. Although chromosome loss is increased in an HR PRR double mutant such as rad51∆ rad18∆ (see further), spontaneous mutation is not synergistically increased [35], so increased translesion synthesis cannot account for the HR PRR double mutant viability. One candidate for a third pathway of damage avoidance is the MGS1 gene, also called WHIP [54–56]. mgs1∆ mutants have no DNA-damage-sensitive phenotype, but have increased homologous recombination rates. The mgs1∆ mutants are synthetically lethal or grow very poorly with additional rad5∆, rad6∆, or rad18∆ mutations [56]. The synthetic lethality does not occur in the mgs1∆ srs2∆ double mutant, further showing that SRS2 has more of a regulatory role than an enzymatic function in the PRR damage avoidance process. A srs2 mutation will suppress the growth defect of a mgs1∆ rad18∆ mutant, presumably by enhancing HR repair of spontaneous damage [56]. Lastly, this rescue of the lethal mgs1∆ rad18∆ double mutant requires functional HR as ablation of HR by a rad51∆ or rad52∆ mutation becomes lethal.

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Table 1 Genomic instability rates Genotype

Wild type

rad51∆

mec1∆ sml1∆

55.5 (27) 77.6 (38) 136 (70) 159 (78) 100 (50)

47.0 (23) 150 (74) 136 (70) 43.6 (22) Not determined

(×106 )

Chromosome loss rates Wild type 2.0 (1) srs2∆ 15.6 (8) rad5∆ 15.0 (7) rad18∆ 8.7 (4) mgs1∆ 3.3 (1.6)

Recombination rates (×105 ) Wild type 1.4 (1) srs2∆ 2.7 (2) rad5∆ 2.3 (1.6) rad18∆ 13.8 (9.9) mgs1∆ 4.3 (3)

1.3 1.9 3.9 4.4 3.1

(0.9) (1.4) (2.8) (3.2) (2)

9.6 (7) 15.4 (11) 32.8 (23) 36.7 (26) Not determined

The numbers in parentheses indicate the fold increase in chromosome loss or recombination rate relative to wild type. Strains used are the same as those listed in the legend to Fig. 4. The mgs1∆ strains were: for mgs1∆ HKY1236-22B × HKY1236-37C and for mgs1∆ rad51∆ HKY1408-1D × HKY1411-1B.

Studies in our laboratory have shown that the mgs1∆ mutant has no increased chromosome loss rate, and a three-fold increase in recombination in homozygous diploids. However, chromosome loss is synergistically increased 50-fold while recombination is only increased 2-fold in mgs1∆ rad51∆ double mutants (Table 1). The RAD51-independent recombination is probably break-induced replication (BIR) [57,58]. BIR is a type of DSB repair where an invading 3 single-strand end primes extended tracts of DNA synthesis, using a homologous sequence as template for strand invasion. Often the invaded sequence is the homologous chromosome. Sequences distal to the DSB are lost, and are replaced by sequences from the invaded donor duplex. BIR is not reciprocal; only one of the DSB ends is repaired and hence only one recombinant product is recovered. The MGS1 gene product is related to replication factor C and probably interacts with the replication machinery during stalled replication to facilitate lesion bypass [59]. It has been proposed to be involved in fork regression or template switching repair synthesis [59], but evidence for a physical interaction with the replication machinery, and more precisely stalled replication complexes, is inferred from association of the human WHIP protein with the WRN protein [60] and genetic interaction of the yeast Mgs1 protein with DNA polymerase ␦ [59]. Nonetheless, the arrest phe-

notype of mgs1∆ PRR double mutants is consistent with a defect in completion of DNA replication [56], and this is strengthened by a genetic interaction of the mgs1∆ mutant with a sgs1∆ mutant [54]. A recent study on tolerance of spontaneous damage in yeast strains multiply deficient in DNA damage repair pathways has revealed overlap in damage repair and a hierarchy in the use of the different pathways to repair damage [61]. Four different damage repair pathways were examined in this study; base excision repair (BER), nucleotide excision repair (NER), translesion synthesis and homologous recombination. Haploid strains that were BER− TLS− REC−, BER− NER− TLS−, or BER− NER− HR− were inviable. In diploids only BER− NER− HR− strains were inviable. These results show that although there is some overlap in repair or tolerance of spontaneous damage, in haploid strains BER is most important while in diploids HR and NER have more prominent roles in damage avoidance or tolerance. It is not know yet whether mgs1∆ PRR double mutant diploids could be viable, rescued by an active intact HR pathway. 1.5. Genomic stability in PRR mutants Most studies of PRR mutant phenotypes have used haploid strains, or diploid strains with specific recombination reporter systems. These studies have suggested that some PRR mutants have increased recombination rates, suggesting that the PRR pathway is first used to repair spontaneous damage through non-recombinational mechanisms. These same studies also contained data suggestive of increases in chromosome loss. If this were true, then this would indicate that HR cannot substitute as a repair mechanism for all spontaneous lesions that are acted upon by the PRR pathway. To better understand the contribution of the PRR processes to maintenance of genomic stability, we have undertaken a study of chromosome loss and recombinational loss of heterozygosity (LOH) in representative PRR mutant diploids. One of the advantages of performing such a study in yeast is that S. cerevisiae is quite tolerant of monosome aneuploidy, so chromosome loss events are stable and easily recovered with a good detection system. Some of those data are presented further, and subsequently discussed in the context of this review on the role of PRR in genomic stability.

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2. Materials and methods 2.1. Yeast strains All yeast strains are derived from W303 and have the basic genotype leu2-3,112 his3-11,15 trp1-1 ura3-1. In addition, one set of strains were ade2-1 hom3-10 can1-100 hxt13 :: KANMX. The other set of strains were ADE2 HOM3 CAN1 HXT13. As is shown in Fig. 3, the HOM3 locus is on one arm of chromosome V, while the CAN1 and HXT13 are on the opposite arm of chromosome V. All strains are isogenic with the exception of the above alleles, and Starting diploid hom3-10

can1-100

HOM3

CAN1

hxt13::KANMX HXT13

Hom+ CanS KanR

CanR segregants 1.

Chromosome loss hom3-10

2.

3.

4.

5.

can1-100

hxt13::KANMX

Chromosome loss and reduplication hom3-10

can1-100

hxt13::KANMX

hom3-10

can1-100

hxt13::KANMX

hom3-10

can1-100

hxt13::KANMX

HOM3

can1-100

hxt13::KANMX

hom3-10

can1-100

hxt13::KANMX

HOM3

can1-100

HXT13

CO/BIR

GC

Chromosome partial deletion hom3-10

can1-100

hxt13::KANMX

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were constructed through crosses and transformation. All mec1 :: TRP1 strains were also sml1 :: HIS3. Each diploid was formed from one parent with the ade2-1 hom3-10 can1-100 hxt13 :: KANMX and the other parent with the ADE2 HOM3 CAN1 HXT13 alleles. 2.2. Determination of chromosome loss, mitotic recombination rates and mutation rates Rates were determined as described [62]. Briefly, fresh zygotes were isolated for each genotype. After growth for 3–4 days at 30 ◦ C on YPD medium, zygotic colonies were resuspended in water. Appropriate dilutions were plated onto complete plates, for measurement of total number of colony forming units, and onto complete plates plus canavanine, to select for canavanine-resistant segregants. These were then replica-plated to complete plates lacking threonine. Colonies that grew on these plates, Canr Hom+ , were counted for determination of recombination rates by fluctuation. Colonies that grew only on the complete plus canavanine plates, Canr Hom− , were counted for determination of chromosome loss rates. Fluctuation tests were determined by the median method [63] and were repeated three to five times for each genotype. Forward mutation rates of CAN1 were determined in haploid CAN1 strains by fluctuation tests [63]. These rates were subtracted from the Canr Hom+ rates, to give recombination rates. In no case was the mutagenesis rate more than 10% of the total Canr Hom+ rate, and in most cases it was less than 1%. 2.3. Other methods PCR reactions on yeast genomic preparations were performed according to standard methods. PCR amplification of the HXT13 locus was performed to distinguish Canr segregants that remained heteroallelic at HXT13, gene conversion events, from Canr recombinants that became homozygous for hxt13 :: KANMX.

HOM3

Fig. 3. Depiction of the chromosome V alleles used in this study and the possible events resulting in Canr segregants. Spontaneous mutation can also result in Canr segregants, but these are not included in the picture as they are rare events in diploid strains compared to the events depicted. The five classes of events can be distinguished by a combination of phenotype analysis, PCR, CHEF gels and tetrad analysis.

3. Results 3.1. Chromosome loss in PRR mutants The studies discussed earlier showed that interchromosomal gene conversion was increased in the

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rad5, rad6, rad18 and srs2 PRR mutants. In some of these papers, loss of heterozygosity at heterozygous recessive markers [33] or loss of heterozygosity at mating type [37] was selected. LOH was increased 70–100-fold in rad18 mutants, showing that the PRR pathway functions to suppress LOH events. These studies did not distinguish between recombination and chromosome loss, both of which could give the LOH phenotype. Therefore, we have examined LOH events in rad5, rad6, rad18 and srs2 mutants. The results show that the PRR genes act differently to suppress LOH. Using strains carrying the chromosome V alleles shown in Fig. 3, we first selected for Canr segregants. We then screened among these for segregants that had also lost heterozygosity at the HOM3 locus and revealed the recessive hom3-10 allele. Two basic types of events result in a Canr Hom− Kanr phenotype; chromosome loss and chromosome loss plus reduplication (events 1 and 2). The remaining phenotype Canr Hom+ Kanr results primarily from recombination events (events 3 and 4). Chromosome deletion in diploids is rare compared to events 1–4. Chromosome loss will result in a diploid cell that is hemizygous for all genes on chromosome V, and upon sporulation and tetrad analysis, will give two viable spores and two dead spores. Chromosome loss followed by reduplication will give a diploid cell that is homozygous for all genes on chromosome V and upon sporulation and tetrad analysis will give four viable spores with identical genotype for chromosome V. srs2∆ mutants had an increased chromosome loss rate of approximately eight-fold above wild type (Fig. 4A and Table 1). rad6∆ mutants did not have an altered chromosome loss rate, and individual fluctuation tests suggested that the chromosome loss rate could be lower than wild type. The wild type rate is close to the limit of accurate detection since loss events are not directly selected for. Rather, they are screened for among Canr LOH events. When the ratio of recombination LOH:chromosome loss LOH is about 100:1 it is difficult to get an accurate ratio of recombination:loss events. Another limiting factor is that chromosome loss events sometimes reduce the growth rate of the diploid cell, so there may be an inherent selection for reduplication events. rad5∆ and rad18∆ mutants had slightly increased chromosome loss rates. Approximately 50% of the

chromosome loss events in rad5∆ and 90% of the chromosome loss events in rad18∆ were reduplication events. These probably result from non-disjunction of the monosomic chromosome V, to revert the cell back to a euploid chromosome number of 32, but it is not clear whether the original event was a loss followed by a reduplication or non-disjunction event, or whether the original event was a non-disjunction of sister chromatids. However, these arose, these loss events turned out to be composed of two identical copies of chromosome V, as determined by tetrad analysis giving four viable spores of identical chromosome V genotype and CHEF gel analysis to show the presence of two copies of chromosome V in the diploid strains that were originally identified as chromosome loss candidates. 3.2. Role of recombination in chromosome loss of PRR mutants We have reported that chromosome loss is increased in rad51∆ diploids [62]. Although sister chromatid exchange is used to bypass replication-blocking DNA lesions [64,65], in diploid cells DNA lesions can also stimulate exchange between homolog chromosomes. The relationship of sister chromatid recombination to homolog recombination in this context is not clear. Models for HR involvement in re-establishing replication forks may involve either strand invasion and exchange followed by Holliday junction resolution, template switching copy-choice mechanisms or fork reversal and restoration [66] (see Fig. 5). The HR gene requirements for these bypass mechanisms are not fully defined. In S. cerevisiae RAD52 is required for Holliday junction formation in DNA replication mutants, but RAD51, which encodes the strand exchange protein Rad51, is not required [67]. Perhaps the replication apparatus leaves the DNA strands in an open conformation and only strand annealing is needed for Holliday junction formation [68]. In these studies Holliday junctions were studied in the repeated rDNA sequence and the repeated nature of the sequence may have allowed some pairing in the absence of the Rad51 protein. It is also not clear whether Holliday junction structures form in wild type cells, or whether they reflect an intermediate only seen in mutant cells. In the yeast S. pombe Holliday junction-containing structures have been observed in association with

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Fig. 4. Rates of chromosome loss in: (A) rad51∆ mutants; (B) mec1∆ mutants. Chromosome loss events from Canr Hom− Kanr segregants were used to determine chromosome loss rates as described in Section 2. Strains used were: for wild type HKY950-12C × HKY1026-1B and HKY953-3A × HKY947-14D; for rad51∆ HKY1039-1A × HKY1038-6C; for srs2∆ HKY1303-1B × HKY1304-2D; for rad5∆ HKY1329-1B × HKY1328-8C, HKY1329-14A × HKY1328-16B, and HKY1329-4B × HKY1328-12B; for rad18∆ HKY1331-16D × HKY1330-12A; for srs2∆ rad51∆ HKY1333-13C × HKY1307-12C; for rad5∆ rad51∆ HKY1362-10C × HKY1362-10D and HKY1372-4B × HKY1373-1A; for rad18∆ rad51∆ HKY1361-6D × HKY1361-4A and HKY1360-2C × HKY1394-13A; for mec1∆ sml1∆ HKY978-9D × HKY986-10D; for srs2∆ mec1∆ sml1∆ HKY1353-2B × HKY1356-34A; for rad5∆ mec1∆ sml1∆ HKY1359-2A × HKY1358-5C and HKY1359-18C × HKY1358-1C; for rad18∆ mec1∆ sml1∆ HKY1350-14C × HKY1349-2B. rad6∆ strains used but not shown in this figure were HKY1329-8D × HKY1328-12D, HKY1329-10C × HKY1328-1C, and HKY1329-13A × HKY1328-5B.

activated origins of replication at unique sequences [69]. Formation of these recombination intermediates is dependent on the HR genes rad22+ , rhp51+ , and rhp54+ , which differs from the requirements for recombination intermediate formation associated with replication in S. cerevisiae. However, rhp51+ is required for efficient origin firing, so it is difficult to separate the genetic requirements for origin fir-

ing from the genetic requirements for formation of Holliday junction-containing structures at activated origins. Formation of Holliday junctions structures in unique replicating sequences was also observed in Physarum polycephalum [70]. Both reports suggest that the joint molecules reflect Holliday junctions between sister chromatids, although it is possible that HR-independent joint molecules are actually

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Fig. 5. Model for lesion bypass by template switching. (A) When replication fork progression is stalled by a lesion (rectangle) on the leading strand template, lagging strand synthesis can continue to create an overshoot nascent strand. Fork reversal allows the nascent sister strands to anneal, providing an undamaged template for extension of the leading strand. Once the leading strand has been extended beyond the stalling lesion, the fork can reform, allowing the nascent strands to pair with the template strands, and allowing synthesis again using the template strands, and bypassing the template strand lesion. (B) When replication fork progression is stalled by a lesion (rectangle) on the leading strand template, lagging strand synthesis can continue to create an overshoot nascent strand. This strand can pair with the nascent leading strand, allowing extension of the leading strand past the stalling lesion. The nascent strands then pair with the template strands and the fork is reformed, bypassing the template strand lesion.

hemicatenanes between the nascent sister chromatids. Alternatively, the Holliday junction that is proposed to result from strand invasion may in fact result from template switching or reversal of the replication fork

to form a four-way junction [31]. Some of the HR events that are regulated by Srs2 may be postreplication repair of ssDNA gaps and thus recombination could occur between homolog chromosomes without

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interfering with replication restart. We do not favor this model because the srs2∆ mutation is lethal in combination with a sgs1∆ mutation, and this is proposed to be due to ssDNA gaps that occur during replication [71]. Moreover, recombination, or more precisely, inappropriate recombination between homologs is thought to be deleterious in srs2 mutants [41,71]. To further understand how chromosome loss originates, we determined whether HR acted independent of PRR in suppression of chromosome loss. Therefore, we examined the chromosome loss rates in rad5∆ rad51∆, rad18∆ rad51∆ and srs2∆ rad51∆ double mutants. We noted that rad5∆ rad51∆ and rad18∆ rad51∆ double mutant diploids had a synergistic increase in chromosome loss (Fig. 4A and Table 1). We interpret this as independent suppression of chromosome loss by PRR and HR. When the PRR error-free pathway is ablated, HR can be used as a back-up mechanism to prevent chromosome loss. The synergistic effect of a HR and PRR double mutant on genomic stability has also been observed in chicken DT40 cells [72]. In our case with yeast, the rad18∆ rad51∆ double mutant was viable, but grew at a slower rate than either single mutant. In contrast, the srs2∆ rad51∆ double mutant diploid did not show a synergistic increase in chromosome loss. Rather, the loss rate in the double mutant appears to be additive. The increase in chromosome loss in the srs2∆ mutant might reflect those events that cannot be acted upon by the PRR pathway to repair damage leading to chromosome retention. This would be consistent with the channeling model for SRS2 of damage repair to the PRR pathway. The synergistic increase in chromosome loss in the rad5∆ rad51∆ and rad18∆ rad51∆ double mutants shows that the active PRR pathway suppresses chromosome loss. Whether SRS2 acts specifically on the RAD5-dependent sub-pathway of the PRR pathway to suppress chromosome loss is not known. SRS2 has been shown to act on the error-free part of the PRR pathway to prevent DNA damage and aid in the bypass repair of replication-blocking DNA lesions [14], and further has been suggested to act only in the RAD5-dependent branch of error-free PRR [16]. The suppression most likely occurs through destabilization of the Rad51 filament on ssDNA [49,50], but whether Srs2 protein has additional activities

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that do not entail an antirecombinase function is not known. 3.3. Role of DNA damage checkpoint functions in chromosome loss of PRR mutants The SRS2 gene shows regulated expression, with expression commencing shortly before S-phase and decreasing at G2 [73]. G2 expression can be induced by DNA damage [73]. The Srs2 protein is phosphorylated after intra-S DNA damage induction by the Mec1, Rad53 and Dun1 checkpoint kinases [74]. These findings suggest that Srs2 activity is regulated, and the protein may be targeted to stalled replication forks by checkpoint functions. To investigate whether spontaneous damage is signaled to checkpoint control of genomic stability, we examined the effects of a MEC1 deletion on PRR mutants. MEC1 encodes a kinase related to ATR of mammalian cells. It is required in both DNA damage and replication stress signaling [75,76]. We have reported that mutants in DNA damage checkpoint genes have elevated chromosome loss rates [62]. In the previous section we reported that PRR mutants have only a modest (three- to eight-fold) increase in chromosome loss rates. Although there is a link between Mec1 and Srs2 in responding to induced DNA damage [74], the srs2∆ mec1∆ double mutant has a synergistic increase in chromosome loss, suggesting that these genes respond in independent ways to spontaneous damage leading to chromosome loss (Fig. 4B). A similar effect was observed in the rad5∆ mec1∆ double mutant (Fig. 4B). The synergistic increase in chromosome loss or genomic instability in double mutants of HR and DNA damage checkpoints has been reported [62,77], although no studies have been reported on DNA damage checkpoint and PRR gene double mutants. An alternative explanation is that, in the mec1∆ mutant, more homologous recombination events occur between homologs. mec1 mutants have been reported to have different effects on mitotic recombination, depending on the event being studied. Kato and Ogawa found no effect on gene conversion, but observed an increase in mitotic interchromosomal recombination [78]. Spontaneous and induced gene conversion was decreased at two loci when heteroallelic intrachromosomal recombination was measured [79]. In another study, gene conversion at the CAN1 locus was unchanged in a

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mec1∆ mutant but other recombination events such as crossing over and BIR were increased seven-fold [62]. Increase in these types of interchromosomal recombination events in the srs2∆ and rad5∆ mutants could lead to inappropriate recombination events that can destabilize chromosomes. In contrast to the above results, the rad18∆ mec1∆ double mutant did not have a synergistic increase in chromosome loss. The loss rate in this double mutant was approximately that of the mec1∆ single mutant. In this mutant SRS2 is still functional, and may act outside of the PRR pathway to repair spontaneous damage, or in some manner facilitate HR when PRR is completely absent. It will be of interest to determine the chromosome loss rate in a srs2∆ mec1∆ rad18∆ triple mutant, to see if SRS2 has an additional function outside of PRR. These findings implicate MEC1 in the RAD5 subpathway of error-free DNA damage avoidance, in the pathway that is suggested to promote template switching for lesion bypass during stalled replication. MEC1, RAD5 and SRS2 may cooperate to bypass damage in a manner that may be an alternative to recombination-promoted replication restart. This is most likely template switching through a copy-choice type of mechanism to bypass lesions on the template strand (Fig. 5). 3.4. Role of HR genes in recombination in PRR mutants As discussed earlier, PRR mutants have elevated recombination levels in gene conversion and direct repeat recombination events. They are also increased in mitotic crossing over. The increased recombination requires the HR functions encoded by RAD51, RAD52, RAD55 and RAD57, depending on the type of recombination event under study [35,42]. To further study the role of PRR genes in repairing spontaneous lesions by a lesion bypass mechanism that is an alternative to recombination, we examined PRR mutants for increases in HR. The system we used, depicted in Fig. 3, involves selection for LOH at CAN1 and then screening among the Canr segregants for those that are not chromosome loss events. These are the Canr Hom+ segregants. The results, shown in Fig. 6A and Table 1, reveal that the srs2∆ mutant has a slight increase in recombination, but not at the fold increase

seen for intrachromosomal recombination [42]. This is probably due to the finding that recombination between homologs in srs2 mutants is often lethal [41]. In another study, ectopic gene conversion, gene conversion between sequences on non-homologous chromosomes in haploid strains, was studied in the srs2∆ mutant [35]. These gene conversions were increased two- to five-fold over wild type, an increase that is not dissimilar from intrachromosomal rates reported for haploid srs2 mutants [43,80]. The PRR mutants rad5∆, rad18∆ and rad6∆ (data not shown) had differing effects on recombination. rad6∆ and rad18∆ mutants were increased approximately 10-fold over wild type for recombination events. This is similar to previous reports that these mutants have increased recombination [33,35–37]. Based on additional tests of the recombination class of events through a combination of genetic analyses and PCR tests for the HXT13 genotype, we find that most of the increase in the recombination fraction is not due to gene conversion events. Instead, increased crossing over or BIR, and more complex events were found (Fig. 3). Interestingly, the rad5∆ mutant did not have a great increase in recombination. This suggests that in the absence of the RAD5 sub-pathway of PRR, lesions can be repaired by an alternative mode that does not involve homolog recombination. Either this is a sister chromatid type of recombination, or is a lesion bypass pathway distinct from the RAD5 sub-pathway. Evidence for a second error-free lesion bypass pathway of PRR has come from double mutant epistasis studies of DNA damage sensitivities [16,18]. At this point there is not a consensus as to the action of SRS2 in the entire error-free branch of PRR versus action in only the RAD5 sub-pathway. If SRS2 is required for recombination mediated by the entire error-free PRR branch, then it should be epistatic to rad5∆, rad6∆, and rad18∆ mutants. This has been found in some studies, but the recombination under study was ectopic gene conversion, not recombination between homolog chromosomes [34,35]. Another complicating factor that may account for the differing results from srs2 epistasis studies for DNA damage sensitivity is that the suppressive phenotype of srs2 mutants seems to depend on the experimental conditions of cell growth. srs2 mutants had suppression activity in logarithmic growth, but not in stationary phase [34]. If Srs2 acts on blocked replication forks,

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1.61E-04

Recombination rate

1.41E-04 1.21E-04 1.01E-04 8.10E-05 6.10E-05 4.10E-05 2.10E-05 1.00E-06

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rad51

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(A) 6.01E-04

Recombination rate

5.01E-04 4.01E-04 3.01E-04 2.01E-04 1.01E-04 1.00E-06

(B)

Fig. 6. Rates of recombination in: (A) rad51∆ mutants; (B) mec1∆ mutants. Chromosome loss events from Canr Hom+ Kanr segregants were used to determine chromosome loss rates as described in Section 2. Strains used were those listed for Fig. 3.

it would be expected to have maximal activity in growing cells. This is also consistent with the cell cycle expression of SRS2 [73]. Rad5 has been proposed to promote copy-choice template switching for lesion bypass [23,38]. This presumably occurs during replication fork stalling. In the absence of Rad5, lesion bypass may occur through HR-mediated sister chromatid exchange. Since sister chromatid exchange does not give a genetically detectable product in our system, rad5∆ mutants will not have increased recombination rates, and thus the rate will not be grossly affected by loss of Rad51. Nonethe-

less, the silent sister chromatid recombination events are necessary to restore replication and maintain chromosome stability, and so the rad5∆ rad51∆ double mutant has a synergistic increase in chromosome loss. Exchange between sister chromatids may be promoted by the Mec1 kinase, so in the rad5∆ mec1∆ more recombination events use the homolog instead of the sister chromatid, giving rise to the rad5∆ mec1∆ increase in homologous recombination and chromosome loss. The increased recombination seen in the rad18∆ mutant is dependent on the HR gene RAD51 (Fig. 6A

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and Table 1). This suggests that most of the increase is due to increased gene conversion or crossing over. The residual events seen in the double mutants srs2∆ rad51∆, rad5∆ rad51∆, and rad18∆ rad51∆ are most likely RAD51-independent events of BIR. 3.5. Role of DNA damage checkpoint functions in recombination in PRR mutants We have found that DNA damage checkpoint mutants combined with HR mutants rad51∆ [62] and rad54∆ (HLK, unpublished observations) have a recombination rate that is only slightly higher than that of the single DNA damage checkpoint mutant. This means that recombination events that occur in the DNA damage checkpoint mutants are for the most part independent of these HR activities. To see if loss of PRR creates substrates for HR that are sensed by the DNA damage checkpoint functions, we examined recombination in srs2∆ mec1∆, rad5∆ mec1∆, and rad18∆ mec1∆ double mutants. As reported previously, the mec1∆ mutant had an increased rate of recombination. Examination of the events occurring in the mec1∆ mutant has shown that gene conversion is not increased over wild type, but crossing over or BIR events are increased seven-fold (HLK, unpublished observations). As discussed, srs2∆ and rad5∆ mutants have modest increases in recombination, whereas the rad18∆ mutant has a substantial increase in recombination (Fig. 6 and Table 1). The srs2∆ mec1∆ double mutant had a multiplicative increase in recombination (Fig. 6B), suggesting that these two genes act independently to suppress recombination, but do not create recombinogenic substrates when mutant. The rad5∆ mec1∆ and rad18∆ mec1∆ double mutants showed increased recombination rates over the rad5∆ and rad18∆ single mutant rates (Fig. 6B and Table 1). Again these results suggest independent action by the PRR pathway and the DNA damage checkpoint functions to prevent recombination events. Since most of the rad18∆-mediated events are dependent on RAD51 (Fig. 6A), it will be of interest to determine whether a rad51∆ mutation will reduce the rad18∆ mec1∆ recombination rate. If this rate is reduced to the mec1∆ rate, this will show that the rad18∆ mutation prevents a different spectrum of recombination events from occurring, compared to those observed in the mec1∆ mutant.

4. Regulation of spontaneous damage repair by the PRR pathway genes The current model for determining whether a lesion is repaired via a branch of the PRR pathway versus homologous recombination involves competition for binding to the ssDNA gap between Rad6/Rad18 complex and Rad51. In actuality, the type of lesion in the ssDNA may determine whether Rad6/Rad18 binds preferentially to the DNA site. UV and MMS lesions are repaired by PRR, but can also stimulate HR, suggesting some overlap in repair of the same substrate. However, cell cycle may also play a role in determining which pathway is used for repair. The cell cycle regulation of SRS2 expression [73] and its modification and induction by DNA damage [74] point to regulated use of PRR versus HR. Whether the same regulation is used for repair of spontaneous damage is not known. The target for Rad51 binding may not be the initial ssDNA gap, but rather the reversed fork with the annealed nascent sister chromatids, which has a DSB end, or a collapsed fork with a DSB end. Phosphorylation of Srs2 in response to HU or MMS treatment partially requires the Mec1 and Rad53 kinases and fully requires the Dun1 kinase [74]. We have examined chromosome loss and recombination in dun1∆ mutants and find no effect on the spontaneous rates. However, another study found a 200-fold increase in gross chromosomal rearrangement rates in haploid dun1∆ strains [81]. Perhaps the level of spontaneous damage in diploid strains is not sufficient to elicit phosphorylation of Srs2. This seems likely as Srs2 is not phosphorylated in haploid strains in the absence of induced damage [74]. However, since chromosome loss is not increased in the dun1∆ diploid while loss is increased in the srs2∆ diploid (Fig. 4 and Table 1), this would suggest that phosphorylation of Srs2 by Dun1 kinase is not necessary for its function in preventing chromosome loss. Alternatively, Srs2 may not be a target of the Dun1 kinase. It has been argued that Srs2 is a direct or indirect target of the Cdk1 kinase and requires an intact Mec1 pathway for its complete function [74]. The synergistic effect on chromosome loss in the srs2∆ mec1∆ mutant suggests that Mec1 kinase affects chromosome stability beyond phosphorylation of Srs2, probably through phosphorylation of many target proteins to stabilize stalled replication forks.

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5. Overlapping damage avoidance pathways

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call this pathway ADA. There are many other damage repair pathways that will not be considered here. The three pathways appear to act independently, but have overlap as strains with mutations in two pathways are more severely affected than single mutants. A summary of the effects on chromosome loss and recombination increases of mutants in these pathways is shown in Fig. 7. The representative mutants shown are mgs1∆ for ADA, rad51∆ for HR, and rad18∆ for PRR.

Studies of double mutant phenotypes, both epistasis studies of damage sensitivity and recombination rates, and synthetic lethal phenotypes, have shown that there are multiple pathways to repair spontaneous damage and prevent LOH. The three pathways to be discussed here are the PRR pathway, the HR pathway and the alternative damage avoidance (ADA) pathway represented by the MGS1 gene. For this discussion we will

Chromosome loss increases

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120 100 78

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10 10 8 6 4

3

3 2

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Fig. 7. Contributions of different repair pathways to genomic stability. The fold increases over wild type rates are shown in ADA− mutant mgs1∆, PRR− mutant rad18∆, HR− mutant rad51∆, HR− PRR− mutant rad51∆ rad18∆, and HR− ADA− mutant rad54∆ mgs1∆. The ADA− PRR− mutant rad18∆ mgs1∆ is inviable. Recombination was measured as described in Section 2.

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Chromosome maintenance seems to be most sensitive to loss of these pathways. ADA and PRR appear to contribute approximately the same degree of chromosome retention, since the ADA HR and PRR HR double mutants have high increases in chromosome loss. Given the ADA PRR double mutant lethality, this suggests that together ADA and PRR perform an essential damage avoidance function. This is probably replication restart at stalled forks. HR contributes an important component since chromosome loss is increased in HR mutants. We would argue that the ADA PRR lethality stems from a chromosome loss rate that is too high to sustain cell growth. Even though HR is intact in the ADA PRR double mutant, its function is not sufficient to prevent lethality. Possibly, not all spontaneous lesions can be repaired by HR in a productive manner. The effect of mutations in these pathways on recombination that gives rise to LOH is more complex. This is in part due to the fact that we are not sure as to the nature of the recombination LOH events. In the HR mutants, these are probably BIR events, but in the ADA and PRR mutants the recombination LOH events could arise from crossing over, gene conversion, or BIR events. Until the nature of the events is clear, the effect on recombination remains a study of a composite nature. Nonetheless, it is clear that recombination events, especially those in the HR mutants, are not increased to the same extent as chromosome loss by additional damage avoidance mutations. The increase in recombination in the HR ADA double mutant represents events that are RAD51-independent. These are presumed to be BIR events. If that is the case, then an active ADA system suppresses them.

6. Further questions regarding maintenance of genomic stability This brief summary of the role of lesion bypass pathways in maintaining genomic stability has raised more questions than it has answered. One of the most important questions relates to the interaction, direct or indirect, between components of these repair pathways and the DNA replication apparatus. Polymerase ␦ mutations have known to interact with the mgs1∆ mutation [56,59] and the PRR pathway is proposed to be used to bypass stalled replication forks. The complex

cascade of PCNA modifications by SUMO and ubiquitin by PRR functions may be involved in the signaling of stalled replication forks [24]. The modifications of PCNA do not target it for degradation. Rather, the different modification of PCNA may determine the type of repair that is used for replication restart. Whether or not PCNA modification is linked to the intra-S checkpoint functions is not known, although UV damage bypass during S-phase involves checkpoints [82] and the Srs2 protein is part of the intra-S DNA damage response [74]. Elucidating the nature of the interaction between the replication complex and these damage avoidance pathways at the molecular level will be an important step in understanding how the damage response and damage avoidance pathways interact. Another important problem is to determine the nature of spontaneous damage that causes genomic instability when a repair pathway is defective. This could be oxidative damage, abasic sites, nicked DNA templates, and limiting DNA precursors. Whether the responses to induced damage or depletion of DNA nucleotide precursors by chemical treatment are identical to responses to spontaneous damage also is not known. PRR mutants have increased spontaneous mitotic recombination rates, measured using both intrachromosomal and interchromosomal recombination detection systems. This observation indicates that substrates that are normally repaired, or damage that is bypassed by the PRR pathway, can be channeled to the HR pathway when PRR is not functional. Presumably some substrates must also be channeled to the ADA pathway as the ADA− PRR− mutant is inviable but the HR− PRR− mutant is viable. Whether some substrates are preferentially acted upon by the ADA system and others by the HR pathway when PRR is not functional is unknown. Lastly, the role of checkpoint signaling and recruitment of repair factors in determining how a lesion is repaired is not known.

Acknowledgements The authors thank Lisa Lisanti, Anastasiya Epshtein and Elena Potylitsina for help in performing some of the cited experiments. We thank Wei Xiao for helpful discussions. This work is supported by grants from the National Institutes of Health.

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