DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae

Mutation Research 486 (2001) 167–184

Mini review

DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae Stacey Broomfield, Todd Hryciw, Wei Xiao∗ Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada S7N 5E5 Accepted 16 April 2001

Abstract DNA postreplication repair (PRR) is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actually removing the replication-blocking lesions. In bacteria such as Escherichia coli, this activity requires RecA and the RecA-mediated SOS response and is accomplished by recombination and mutagenic translesion DNA synthesis. Eukaryotic cells appear to share similar DNA damage tolerance pathways; however, some enzymes required for PRR in eukaryotes are rather different from those of prokaryotes. In the yeast Saccharomyces cerevisiae, PRR is centrally controlled by RAD6 and RAD18, whose products form a stable complex with single-stranded DNA-binding, ATPase and ubiquitin-conjugating activities. PRR can be further divided into translesion DNA synthesis and error-free modes, the exact molecular events of which are largely unknown. This error-free PRR is analogous to DNA damage-avoidance as defined in mammalian cells, which relies on recombination processes. Two possible mechanisms by which recombination participate in PRR to resolve the stalled replication folk are discussed. Recombination and PRR are also genetically regulated by a DNA helicase and are coupled to the cell-cycle. The PRR processes appear to be highly conserved within eukaryotes, from yeast to human. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Saccharomyces cerevisiae; Postreplication repair; Mutagenesis; Recombination; Cell-cycle regulation

1. Introduction Although DNA has been selected during evolution as a major carrier of genetic information due to its relative stability over other macromolecules such as RNA, it is by no means inert. The reactivity of DNA with endogenous or environmental agents results in genetic modifications, which can have mutagenic or lethal effect upon replication. Many of the lethal replication-blocking lesions are typically repaired by ∗ Corresponding author. Tel.: +1-306-966-4308; fax: +1-306-966-4311. E-mail address: [email protected] (W. Xiao).

nucleotide excision repair (NER) and base excision repair pathways. If these pathways are saturated or unable to repair such lesions prior to the onset of S phase, cell death could result. To prevent cell death in such circumstances, all cells contain DNA damage tolerance pathways. DNA damage tolerance acts to reinitiate replication in the presence of damage, without lesion removal [1]. The “better safe than sorry” philosophy has apparently been adapted by all organisms and the genetic network highly conserved throughout evolution. The mechanism could also be important in an environment with new chemical threats, since they may cause lesions that existing repair pathways are unable to handle.

0921-8777/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 ( 0 1 ) 0 0 0 9 1 - X

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UV-induced DNA lesions are usually repaired by NER, but if this pathway is not functional, these lesions will persist into S phase and interfere with DNA replication. This is manifested in the appearance of single-stranded DNA breaks, which can be observed through separation of genomic DNA in an alkaline sucrose gradient [2–5]. After a short incubation period the fragmented genomic DNA converts to larger molecular weight species, similar to the DNA of unirradiated controls [2–5]. This restoration process is defined as postreplication repair (PRR). The low molecular weight DNA detected using alkaline sucrose gradients is assumed to arise from stalled replication forks creating areas of single-stranded gaps [2,4,5]. The lesions responsible for stalling replication are not removed from the DNA during this process, and persist even after the gaps are resolved [6,7]. If replication-stalling lesions are recognized by the excision repair pathways, this damage might be repaired before the next round of DNA replication.

2. DNA postreplication repair and mutagenesis in Escherichia coli The DNA damage tolerance activity in E. coli is encoded by genes that are part of the SOS regulon and induced in response to regions of single-stranded genomic DNA [8]. The two SOS-dependent mechanisms of DNA damage tolerance are recombinational bypass and translesion DNA synthesis (TLS). Recombination-based bypass is the predominant form of PRR in E. coli [9], although TLS has received much more attention recently. RecA is involved in homologous recombination and the SOS tolerance response. Within PRR, RecA has both a regulatory role and a mechanistic role; it is responsible for the activation of the SOS response, and is absolutely necessary for facilitating both recombination-mediated gap filling and TLS [9]. In recombination-mediated PRR, RecA and the RecFOR complex promote resolution of the stalled replication fork by allowing damage bypass via template-switching. After daughter strand synthesis is interrupted, RecA and RecFOR facilitate the continuation of leading strand synthesis from a homologous chromosome template. The template-switching event allows the sequence containing the thymine dimer

to base-pair with the displaced sequence from the homologous chromosome. Resolution of the recombinational structures results in complete gap filling (reviewed in [10,11]). TLS is facilitated by the mutagenic DNA polymerases DinB and UmuD2 C, both encoded by genes belonging to the SOS regulon. DinB (PolIV) was shown to have low processivity, no proofreading activity, and an ability to elongate misaligned template/primer structures, inducing −1 frameshift mutations [12]. The UmuD2 C complex, named PolV, is an error-prone DNA polymerase on both damaged and undamaged DNA templates [13,14]. PolV bypasses abasic sites roughly 100× more efficiently than the replicative polymerase PolIII, preferentially inserting A across from the abasic site [13,14]. As both PolIII and PolV are required for the error-prone “repair” of a gapped plasmid bearing an abasic site [15], it is hypothesized that after replicating over the lesion, PolV dissociates from the template, allowing PolIII to complete DNA replication [14]. The SOS regulon is an elegant example of how the bacterial cell can regulate DNA damage tolerance. After sustaining moderate levels of DNA damage, the cell initially up-regulates an error-free mode of damage-avoidance mediated by template-switching. Under massive genomic stress, a stronger SOS response induces transcriptional up-regulation of umuDC and dinB and translational modification of UmuD, thereby preventing cell death and providing an option for environmental adaptation under genotoxic stress conditions. Please refer to other recent reviews [11,16,17] for more detailed mechanisms of DNA damage tolerance in E. coli and other prokaryotic organisms.

3. Genes involved in PRR and mutagenesis in Saccharomyces cerevisiae As previously mentioned, S. cerevisiae and E. coli share the PRR phenomenon whereby after UV irradiation there are discontinuities in DNA replication, creating single-stranded gaps [4,5]. To identify genes responsible for PRR in yeast, alkaline sucrose gradients were used to examine the ability of various yeast mutants to resolve the single-stranded gaps created after UV irradiation [4,5]. The mutants examined

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were selected based on their extreme sensitivity to a broad spectrum of DNA damaging agents, their independence from other known DNA repair pathways, or their analogous activities to known SOS regulon proteins. These studies determined that PRR in S. cerevisiae is dependent upon RAD6 and RAD18, and partially on RAD52, whereas the rev3 mutant does not exhibit PRR defect [4]. Despite the identification of PRR-specific genes, many questions regarding eukaryotic PRR remain to be addressed. For example, it is not currently known what occurs upon replication-stalling. The single-stranded gaps detected in the alkaline sucrose gradient may arise from replication restart further downstream of the replication-blocking lesion or, rather than reinitiating stalled replication, it is possible that downstream synthesis may be completed by the neighboring replicon [5]. It is also not clear what mechanism is responsible for gap filling: a translesion bypass mode of replication, or a recombination-mediated template-switching event that allows the cell to replicate past the damage. Another question left unanswered is how cells control the switch between mutagenesis and error-free PRR. A closer look at the putative members of PRR in S. cerevisiae may help elucidate these questions. 3.1. RAD6 and RAD18: founding members in PRR Both rad6 and rad18 were isolated in a screen to identify UV-sensitive S. cerevisiae mutants [18]. RAD6 defines one of three radiation repair epistasis groups, to which RAD18 belongs. Further analyses showed that both rad6 and rad18 mutants were sensitive to a wide range of DNA damaging agents and defective in PRR activity [4,5]. Prior to any biochemical studies on Rad6, it was observed that rad6 cells displayed increased spontaneous mutagenesis and a loss of UV-induced mutagenesis [19–21]. These initial results led to the conclusion that the RAD6 pathway promoted damage-induced mutagenesis [20]. It was later confirmed that the mutagenic response was indeed dependent upon RAD6 and RAD18 [22,23]. Rad6 is one of 13 ubiquitin-conjugating enzymes (Ubcs) in S. cerevisiae and is involved in diverse cellular functions. The rad6 null (rad6
) mutants are defective in PRR, resulting in an extreme sensitivity to a wide variety of DNA damaging agents and a defect in induced mutagenesis [24]. RAD6 is also

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necessary for sporulation [25], telomere silencing [26], and protein degradation based on the amino-end rule (N-end rule) [27]. Ubcs are an essential component of the ubiquitination pathway. Through consecutive reactions of ubiquitin activation (E1), conjugation (E2 or Ubc), and ligation (E3), the 76 amino acid ubiquitin (Ub) is covalently linked to target proteins for functional modification, for cellular signaling, and as a degradation signal [28]. All cellular functions catalyzed by Rad6 require its Ubc activity, since substitution of the active site cysteine (Cys88) residue in Rad6 (Ubc2) confers the rad6
phenotypes [29]. Given that protein degradation is a major function of the ubiquitination pathway, it is not surprising that rad6 mutants are defective in protein degradation via the N-end rule [27]. However, how the Ubc activity of Rad6 mediates PRR, sporulation and telomere silencing is still not clear. The role of Rad6 is perplexing: how can a single activity be modulated to take part in such diverse pathways? Construction of specific mutations and biochemical analyses helped discern regions of the protein required for different cellular functions. The extremely acidic carboxyl-terminal tail (20 out of 23 amino acids are acidic) of Rad6 mediates the ubiquitination of histone H2B [30,31], which is required for sporulation [25]. The N-terminus of Rad6 is involved in N-end rule protein degradation [27] and sporulation [25]. Removal of the N-terminus of Rad6 (rad6
1–9 ) does not disrupt E2–Ub thiolester formation, but prevents the interaction between Rad6 and an E3 protein, Ubr1 [32], which is required for N-end rule protein degradation [27,33]. The rad6
1–9 mutation confers a moderate UV-sensitivity as compared to the extreme sensitivity of the rad6
mutant, and the rad6
1–9 mutant is proficient in UV-induced mutagenesis [32]. Hence, in addition to its roles in protein degradation and sporulation, it appears that the N-terminus of Rad6 may be involved in an activity specific for error-free PRR (discussed in Section 3.3). Unlike Rad6, Rad18 is only involved in PRR. Due to the PRR defect, both rad6
and rad18
are sensitive to UV, X-rays, ␥-rays, 4-nitroquinoline-N-oxide (4NQO), trimethoprim, bleomycin, and mono- and bifunctional alkylating agents [9]. Furthermore, rad18 cells are defective in the repair of single-stranded gaps created by ␥-rays, but are proficient in repairing double-stranded breaks created during the same treat-

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ment [34]. Rad18 is also involved in maintaining the integrity of single-stranded DNA (ssDNA) and linear DNA created after a DNA damaging treatment [35]. This is evident by the increased DNA strand breaks after ␥-irradiation of rad18 cells. The degradation observed is not dependent on DNA replication, nor is it due to cell death [35]. While the genetic interaction between RAD6 and RAD18 was established by epistasis analysis, coimmunoprecipitation studies provided direct evidence for a biochemical interaction via the formation of a Rad6–Rad18 heterodimer [36], which is required for PRR [37]. The complex possesses a Ubc activity, a ssDNA-binding activity and an ATPase activity [38]. The postulated function of the Rad6–Rad18 heterodimer is similar to that of RecA in E. coli: Rad18 binds single-stranded DNA formed at a stalled replication fork and targets Rad6 to the site of damage [37,38]. Neither Rad6 nor Rad18 shares homology with E. coli SOS proteins, nor do they possess the enzymatic activities required to resolve the ssDNA formed upon replication-stalling. It has not been determined how these two proteins, unique to eukaryotes, provide an activity analogous to E. coli PRR. Hence, identification of Rad6–Rad18 targets or downstream events becomes crucial to better understand the mechanisms of PRR. 3.2. Translesion DNA synthesis Although the mechanisms associated with the Rad6–Rad18 heterodimer are not fully understood, the requirement of RAD6 and RAD18 for damage-induced mutagenesis indicates that mutagenic bypass is one of the tolerance mechanisms, and recent studies confirm that TLS of replication blocks is employed to tolerate DNA damage. This activity is carried out by special non-essential DNA polymerases, providing yeast with an activity analogous to E. coli SOS mutagenesis. The biochemical activities of these non-essential mutagenic polymerases have been the subject of many recent reviews [16,17,39,40]. This review incorporates the subject of TLS only in the context of the entire PRR process. UV radiation is known to be mutagenic, and prior to 1996 it was presumed to be a consequence of an active DNA repair/synthesis process. To study this mutagenic process in yeast, Lemontt [41] isolated

S. cerevisiae mutants incapable of reverting the arg4-17 and lys1-1 alleles in response to UV irradiation. These reversionless mutations (rev1, rev2, and rev3) rendered cells moderately UV-sensitive, indicating that mutagenesis was indeed a damage tolerance mechanism. It was soon realized that other known UV-sensitive mutants might also display this reversionless phenotype. Prakash [21] screened a panel of rad mutants and found that rad6 and rad9 cells were defective for the chemically-induced reversion of cyc1-131. Genetic evidence pointing to the possibility of a common mutageneic pathway in S. cerevisiae came from the finding that rad6-1 was epistatic to rev3-1 for UV-sensitivity [20]. Isolation of other mutagenesis-defective mutants soon followed. Lemontt [42,43] isolated the ultraviolet mutation resistant umr1, umr2 and umr3 mutants, which exhibited decreased UV-induced CanR forward mutations, as well as UV-induced reversions. The rev4, rev5, and rev6 isolation was based on a decreased UV-induced reversion of the frameshift allele his4-38 [44], and rev7 was defective in the UV-induced reversion of a lys2 allele [45]. The effect of the rev7-1 mutation was found to be target allele-specific and mutagen-specific [45]. The ngm2-1, defective in MNNG-induced reversion of his1-7 [46], also had an allele-specific effect, as it inhibited induced reversion of his1-7, ilv1-92, and UV-induced CanR , but had no effect on induced cyc1-115 and his4-38 [46]. The genetic relationship between REV4, 5, 6, NGM2 and the Rad6 pathway is currently unknown. REV3 encodes a 173 kDa protein with conserved DNA polymerase motifs [47]. Biochemical studies have shown that Rev3 and Rev7 dimerize to form DNA polymerase ␨ [48]. Purified Pol␨ can replicate over a cis-syn cyclobutane thymine (T–T) dimer on an oligonucleotide template at least 10 times more effectively than Pol␣, but the enzyme has a low processivity. Rev1 has a deoxycytidyl transferase activity; it transfers a single dCMP to the 3 end of a DNA primer in a template-dependent reaction [49]. However, Rev1 cannot insert a C across from a T–T dimer in vitro [48]. This suggests that perhaps Rev1 is used for replication across (6-4) photoproducts instead. In vivo, Rajpal et al. [50] showed that Rev1 is required for all Pol␨-dependent UV-reversion of arg4-17. This appears to be inconsistent with an in vitro observation in which purified Pol␨ alone can

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bypass a T–T dimer [48]. However, when interpreting in vitro results one must bear in mind that what is observed “in the test tube” is not necessarily an accurate reflection of events in vivo, where chromosomal DNA is the replication template, and the entire replication apparatus is present. An alternative explanation would be that the requirement of Rev1 is target allele-specific and lesion-specific. The apurinic/apyrimidinic endonuclease encoded by APN1 in S. cerevisiae is required for base excision repair of alkylated and oxidatively damaged DNA bases. An abasic lesion is non-informative for the replicating polymerase, and poses a block to DNA replication [51,52]. The apn1
mutant has a mutator phenotype [53,54], indicating that TLS might be used to replicate over abasic sites. It was shown that Rev1 inserts a C across from an abasic site in vitro, and DNA Pol␨ extends this primer, implicating both Rev1 and Rev3 in abasic site-induced mutagenesis [49]. The requirement for both Rev1 and Pol␨ in abasic site bypass was demonstrated in vivo by the observation that introduction of a rev1
, rev3
or rev7
mutation into an apn1
apn2
strain eliminated all methyl methanesulfonate (MMS)-induced CanR mutations [51]. Thus, it appears that for both UV- and abasic site-induced mutagenesis, Rev1 might insert the first nucleotide at the replication block and enable Pol␨ to replicate over the offending lesion. While S. cerevisiae appears to employ the novel Pol␨ as its major TLS activity, it does contain two UmuC and DinB homologs; one, the above-mentioned Rev1 [55], and another, Rad30, was discovered by searching the Saccharomyces Genome Database for DinB-like sequences. Like dinB, RAD30 is UV-inducible [56,57]. The rad30
mutant is sensitive to killing by UV [56,57], with minor sensitivities to other DNA damaging agents [57]. RAD30 has been placed in the RAD6 epistasis group by virtue of rad6
and rad18
being epistatic to rad30
for UV-sensitivity [56]. As rad30
was additive to rev1
, rev3
, and rev7
for UV-sensitivity, and the rad30
mutant displayed no defect in UV-induced reversion of trp1-1, RAD30 was placed in the error-free arm of PRR [56]. Rad30 is a newly discovered DNA polymerase (Pol␩) that has low fidelity on an undamaged template in vitro, but a high fidelity over T–T dimers [58]. Unlike Pol␦, which arrests before the T–T dimer, Pol␩ inserts A’s across from both T’s

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[58]. Thus, Pol␩ is required for error-free TLS after UV-induced DNA damage. It was further shown that the Rad30 DNA polymerase activity is required for both resistance to killing and prevention of mutagenesis in response to the UV irradiation of yeast cells [59]. Not only is Pol␩ required for error-free TLS of UV-induced DNA damage, but it is also involved in N-methyl-N -nitro-N-nitrosoguanidine (MNNG)induced mutagenesis. Thus, Pol␩ is either error-prone or error-free, depending upon the DNA lesion encountered. For example, a rad30
pol32
double mutant abolishes MNNG-induced can1R forward mutagenesis [60]. Pol32 is a non-essential subunit of the Pol␦ complex [61]. Unlike pol32
, which is partially defective in this form of mutagenesis, the rad30
single mutant shows no defect, and it was therefore suggested that Pol␦ is more error-prone in its bypass of MNNG-induced lesions than is Pol␩. Pol␩ was 10 times more efficient at bypassing O6 -methylguanine (O6 -MeG) in vitro than was Pol␦. Pol␦ has strong stall sites both immediately preceding the lesion, and directly across from it, while these stall sites were much weaker for Pol␩ [60]. Pol␩ inserts a C across from O6 -MeG twice as frequently as does Pol␦, although both Pol␩ and Pol␦ can be error-prone. Pol␦ is a replicative polymerase and may therefore encounter O6 -MeG lesions during S phase. If Pol␦ fails to bypass O6 -MeG, it is possible that Pol␩ will be recruited to facilitate TLS [60]. In summary, it appears that different TLS polymerases are required to bypass different DNA lesions, as a rev1
, rev3
or rev7
mutation abolishes abasic site-induced forward mutation [51], whereas the pol32
rad30
double mutant abolishes O6 -MeG-induced forward mutation [60]. The unanswered questions are: how does the Rad6 pathway regulates this polymerase switching; and how is it decided which polymerase is used at a given lesion and at a given cell-cycle stage? The idea that Pol␦ is replaced by a TLS polymerase in order to bypass a replication-blocking lesion, and that this new polymerase is in turn replaced by the replicative polymerase in order to complete DNA replication implies the involvement of Pol␦ in TLS. One would expect to find alleles of POL3 that are defective in damage-induced mutagenesis. This is indeed the case in S. cerevisiae. Giot et al. [62] isolated pol3-13, a UV-, ␥-, and MMS-sensitive and temperature-sensitive allele of POL3. They observed

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that for UV-sensitivity, both rev3
and rad18
are epistatic to pol3-13, and rad18
partially rescues the temperature-sensitive phenotype of pol3-13, implying that Rad18 is detrimental to DNA replication in the pol3-13 mutant. Interestingly, while pol3-13 actually elevates spontaneous CanR mutations, it eliminates all UV-induced mutagenesis at the same locus. Together, these results support the contention that Pol␦ is required for the TLS of replication-blocking DNA damage. 3.3. Error-free PRR Although TLS in yeast provides an important tolerance activity, it is not the only, or even preferred mechanism utilized by Rad6-dependent PRR. Early experiments provide at least three lines of evidence to support the existence of another Rad6-dependent and error-free PRR pathway: (1) there is a discrepancy between the extreme DNA damage sensitivity of rad6 and rad18 mutants compared to the moderate sensitivity of rev3 mutants; (2) in the PRR assay rev3 mutants did not display a PRR defect [4,5], further supporting the observation that mutagenesis was secondary to a tolerance process involving sister chromatid exchange (SCE) [63]; (3) the rad6
1–9 mutation appears to separate certain levels of UV-sensitivity from UV-induced mutagenesis [32], suggesting that RAD6 controls at least two separate subpathways. To further understand DNA damage tolerance, identification and characterization of the genes and mechanisms comprising error-free PRR is required. Several newly identified mutants display the DNA damage sensitivity and mutagenesis phenotypes characteristic of error-free PRR-deficient cells. Epistasis analysis of mms2
[64], ubc13
[65,66], rad5
[67] and an allele of POL30 [68] has placed the corresponding genes in the error-free division of PRR. The mms2-1 mutant was isolated by its sensitivity to MMS as part of a screen to identify genes responsible for the repair of DNA alkylation damage [69]. The corresponding MMS2 gene was isolated and studies indicate that although Mms2 shares strong homology with most Ubcs, it does not possess a Ubc activity [64]. Unlike the original mms2-1 mutant, which is only sensitive to MMS, the mms2
mutant is moderately sensitive to MMS, UV [64], ␥-rays [70] and other DNA damaging agents (Broomfield and Xiao,

unpublished results). This broad spectrum of damage sensitivity is reminiscent of the Ubc-defective ubc2/rad6 mutant. This speculation was solidified after the observation that rad6
and rad18
mutations are indeed epistatic to mms2
for UV- and MMS-sensitivity, suggesting that MMS2 functions in the RAD6 pathway. Interestingly, the mms2
mutation increases both spontaneous and UV-induced mutagenesis in a REV3-dependent manner and is synergistic with rev3
for UV- and MMS-sensitivity. Based on these observations, it was proposed that MMS2 plays a role in error-free PRR, which is parallel to REV3-mediated mutagenesis [64]. The observation that rad6
1–9 is epistatic to mms2 led to the hypothesis that Mms2 may be an accessory protein and modulate the activity of Rad6 [64]. The roles of Mms2 in other Rad6-mediated activities were pursued; the mms2
mutation causes a moderate defect in sporulation, has a minor effect on protein degradation, and no effect on telomeric silencing [71]. Hence, Mms2 appears to be specific for error-free PRR, but may also affect other Rad6-mediated processes. Vigorous in vivo and in vitro experiments failed to demonstrate that Mms2 directly interacts with Rad6/Ubc2 (Xiao et al., unpublished observations). It turned out that Mms2 forms a stable complex with a novel Ubc, Ubc13, which is required for in vitro di-Ub formation via Lys-63 [65] instead of the conventional Lys-48 chain assembly mediated by most other Ubcs [28]. Genetically, ubc13 and mms2 are epistatic to each other, and to the UbK63R mutation resulting in a ubiquitin Lys63 → Arg63 substitution [72], with respect to killing by UV [65]. Additional genetic analyses confirm that UBC13 is a member of error-free PRR and plays an equivalent role in PRR with MMS2 [66]. Despite the fact that both Ubc13–Mms2 [64,65] and UbK63 [72] are involved in PRR and that mutations in these genes confer comparable sensitivity to DNA damaging agents, there exists a clear discrepancy: mms2
[64] and ubc13
[66] are proficient in UV-induced mutagenesis, whereas the UbK63R mutant is defective in such an activity [72]. This discrepancy may be explained by the fact that Ubc13–Mms2 is not the only cellular Ubc to catalyze UbK63 assembly; polyubiquitination of the ribosomal protein L28 via UbK63 is independent of Ubc13 and Mms2 [73]. Identifying the cellular targets of the Ubc13–Mms2

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complex, including any possible E3 affiliation, would be key to furthering our understanding of error-free PRR. Within the RAD6 pathway, RAD5 has been assigned to the error-free branch based on the observations that rad5 was synergistic with rev3 for killing by UV and did not affect UV-induced mutagenesis [67]. It was concluded [67] that although rev2-1 is allelic to RAD5, the initial isolation of rev2-1 for its reversionless phenotype was possibly an artifact of the allele, since the reduced UV-induced reversion was specific for the arg4-17 ochre mutation [41]. Deletion of RAD5 confers a moderate increase in spontaneous mutagenesis, a dramatic elevation in simple repeat stability [67], and enhanced non-homologous end-joining for the repair of DSBs [74]. The rad5
and rad18
are hyper-recombinant, and the level of recombination of the rad5
rad18
double mutant does not exceed levels seen in either single mutant [75]. Furthermore, the hyper-recombinant phenotype in rad5
and rad18
cells is dependent on the RAD52 pathway [75], indicating that Rad5 and Rad18 may prevent certain forms of recombination that are detrimental for DNA damage tolerance or that these mutants accumulate recombinogenic structures. RAD5 encodes a 134 kDa ssDNA-dependent ATPase [67,76]. Despite the presence of conserved helicase motifs, Rad5 does not possess a helicase activity [76]. Recently, it was shown that the RING finger motif of Rad5 is required for binding Ubc13 [77]. Two-hybrid analysis and coimmunoprecipitation studies led to the conclusion that Rad5 can homodimerize as well as heterodimerize with Rad18 [77]. Furthermore, the Rad5–Rad18 physical interaction brings two Ubc complexes to chromatin in response to DNA damage, which leads to a model of multivalent assembly of PRR proteins at the damage site [77]. Based on analogy with the E. coli PRR model and genetic evidence, it is expected that a recombination– replication process would be required for the error-free mode of DNA damage tolerance. The participation of DNA polymerase machinery in error-free PRR is illustrated through characterization of a POL30 mutant allele, as discussed below. Genetic interactions between PRR and recombination will be discussed in Section 4. Using double alanine scanning mutagenesis, Ayyagari et al. [78] isolated pol30 mutants with various phenotypes. The pol30-46 mutation is especially

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interesting since it renders cells sensitive to UV and MMS and does not appear to cause a defect in replication [78]. The DNA repair defect of this mutant was associated with the RAD6 pathway, since rad18
and rad6
are epistatic to this allele. Furthermore, pol30-46 does not affect UV-induced mutagenesis and is synergistic with rev3
in response to UV-induced killing [68]. These observations strongly indicate a role for POL30 in the error-free mode of PRR. In an alkaline sucrose gradient assay, UV irradiated pol30-46 mutants show increased fragmentation of genomic DNA compared to the wild type, even after a recovery period [68]. Pol30 is involved in many cellular activities. It is necessary for both Pol␦ (Pol3–Pol31–Pol32) and Polε (Pol2) during DNA replication, and is further required for cell-cycle control, NER and mismatch repair [79]. To determine whether one of the Pol30-associated polymerases were also involved in error-free PRR, temperature-sensitive mutants were used to assess the involvement of both polymerases in PRR. At the non-permissive temperature, the pol2-18 mutant did not display a defect in PRR in the alkaline sucrose gradient assay, suggesting that Polε is not involved in error-free PRR [80]. In contrast, the pol3-3 mutation strongly reduced the PRR activity, suggesting that Pol␦ is probably responsible for the error-free mode of DNA synthesis [80]. Unfortunately, unlike pol30-46, an allele-specific POL3 mutant defective in error-free PRR has not been reported, although pol3-13 is defective in both damage-induced mutagenesis and damage-induced recombination [62]. The discovery of genes implicated in error-free PRR led to the investigation of genetic interactions among these genes. Epistasis analyses with respect to MMS- and UV-sensitivity revealed a synergistic interaction between rad5
and pol30-46, whereas mms2
is additive to both rad5
and pol30-46 [81]. These observations provide further evidence as to the complexity of PRR, whereby the individual error-free PRR proteins may function in separate but coordinated processes. A two-subpathway hypothesis for error-free PRR evolved from the observed genetic interactions; these two subpathways are mediated separately by RAD5 and POL30/POL3, with MMS2 and UBC13 promoting both subpathways (Fig. 1). It was further argued that since the rad5
pol30-46 double mutant is less sensitive to UV and MMS than the rad18


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Fig. 1. A proposed model of PRR within S. cerevisiae. Apart from the independent mutagenic processes of PRR, there appears to be two separate error-free PRR processes represented by Rad5 and Pol␦-PCNA. This figure is adapted from [81].

single mutant, but the rad5
pol30-46 rev3
triple mutant is as sensitive as rad18
, together the rad5
, pol30-46, and rev3
mutations eliminate all aspects of PRR [81] (Fig. 1). Although the above model is attractive and consistent with most of previous observations, it is challenged by a recent conflicting report [77] where rad5
was found to be epistatic to both mms2
and ubc13
with respect to UV-sensitivity. Although the discrepancy may reflect different strain backgrounds, the issue has to be resolved to establish a more reliable model. Together while these two recent publications [77,81] have organized the genes of error-free PRR, they fail to address the possible downstream molecular event(s) leading to DNA damage tolerance. In the next section, we focus on the potential roles of recombination proteins in error-free PRR.

4. Cooperation between PRR and recombination for DNA damage tolerance It is not disputed that Rad6 and Rad18 are necessary for both the error-free PRR and TLS aspects of PRR in S. cerevisiae. While TLS is well defined as a series of DNA polymerase activities across the otherwise replication-blocking lesion, error-free PRR responsible for the resolution of the stalled replication fork

cannot be exclusively performed by the Rad6–Rad18 and Rad5–Ubc13–Mms2 complexes. There is a large body of evidence suggesting that recombination is involved in, and cooperates with, PRR to achieve DNA damage tolerance. In this aspect, we prefer a phrase “damage-avoidance” over error-free PRR because through a recombination-mediated event, the DNA polymerase avoids replicating over the offending lesion. 4.1. SRS2: a molecular switch between PRR and recombination? As previously mentioned, the rad6 and rad18 mutants are extremely sensitive to UV and other DNA damaging agents. Suppressors (radH/srs2) of the UV-sensitivity of both mutants were isolated from separate screens [82–84] in order to uncover novel genes involved in the PRR pathway. Surprisingly, all the above suppressor mutations are allelic to the SRS2 gene. In an independent screen, srs2 was isolated as an allele responsible for an increased rate of gene conversion (SRS2 = HPR5 [85]). The cloned SRS2 encodes a DNA helicase with 3 –5 polarity [86]. Three lines of evidence support the hypothesis that Srs2 channels lesions into the Rad6-dependent DNA damage tolerance pathway, preventing recombination

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repair from inappropriately resolving stalled replication forks. First, the srs2 mutation will only suppress the extreme UV-sensitivity of rad6 or rad18 if there is a functional recombination repair pathway, since mutations in RAD51, -52, -54, -55 or -57 result in phenotypes similar to the single rad6 or rad18 mutants with respect to UV-induced killing [84,87]. Thus, it is assumed that the extreme UV- and MMS-sensitivities caused by rad6 and rad18 mutations are partially due to the Srs2-mediated inhibition of recombination repair. The second piece of supporting data pertains to the phenotype of the srs2 single mutant. The srs2 cells display a moderate UV- and MMS-sensitivity; suppressors of this sensitivity are all allelic to RAD51 [88,89]. It was assumed that removal of Rad51 in an srs2 background prevents the potential use of homologous recombination, thus preventing unfavorable recombination events. It is further predicted that the stalled replication fork is not resolved preferentially by recombination and attempts at using

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a Rad51-dependent recombination mechanism to resolve such a structure may result in cell death. A third supporting observation is that srs2 cells have elevated levels of recombination [85,90,91]. It is argued that in the absence of SRS2 function, cells promote homologous recombination [85]. Studies pertaining to SRS2, RAD6, RAD18 and the genes of the recombination repair pathway have provided a genetic framework for the organization of PRR, but they do not address whether Srs2 inhibits recombination or actually promotes PRR. Results from our laboratory show that srs2
is epistatic to mutations in all members of PRR and mutagenesis, including rad6
, rad18
, mms2
, rev3
, rad30
, pol30-46, and rad5
[71]. Although srs2
does not affect spontaneous and DNA damage-induced mutagenesis, it completely abolishes the elevated mutagenesis in the mms2
mutant (Broomfield and Xiao, in preparation). Based on these observations, we propose that Srs2 is required to initiate PRR (Fig. 2).

Fig. 2. Srs2 controls PRR and DNA recombination repair in S. cerevisiae. (A) In wild type cells, Srs2 promotes both Rad6-dependent error-free and error-free modes of PRR. (B) In srs2
cells, only Rad52-dependent recombinational processes are utilized to tolerate replication-blocking lesions regardless of the presence or absence of Rad6 and Rad18. In this manner, srs2
results in an intermediate level of sensitivity to killing by a wide range of DNA damaging agents but suppresses the DNA damage sensitivities of rad6
and rad18
cells. (C) With a functional Srs2, Rad52-dependent recombination is inhibited, rendering rad6
and rad18
cells extremely sensitive to DNA damaging agents.

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Evidence for Srs2 promoting Rad6-mediated PRR is phenotypic, not biochemical. There are at least two alternative hypotheses on how Srs2 helicase acts in this capacity. At the stalled replication fork, Srs2 may catalyze the unwinding of nascent DNA, creating a template for Rad6-mediated PRR. Alternatively, Srs2 may bind to a DNA structure within the replication bubble and, via protein–protein interactions, preferentially initiate the Rad6 pathway. Until more biochemical data is obtained, the molecular process by which Srs2 promotes PRR remains speculative. 4.2. Recombination repair and replication restart While isolation of the srs2 mutants suggests a distinction between PRR and the conventional recombination repair pathways, it does not rule out a role for recombination enzymes to assist PRR-mediated replication processes. As a matter of fact, a number of observations from different organisms argue for recombination-mediated replication restart as a unified mode of a major DNA damage tolerance mechanism. In E. coli, recombination-based replication restart represents the error-free mode of DNA damage tolerance. In S. cerevisiae, cells deficient in NER tolerate replication-blocking lesions mainly by employing a Rad52-dependent SCE event [92]. In mammalian cells, DNA damage-induced Holliday structures were observed in electron micrographs of DNA isolated from MMS-treated cells [93]. Early studies indicated a role for recombination in tolerating UV-induced DNA damage in uvr E. coli mutants. Density transfer experiments showed that recombinants formed between irradiated DNA strands and strands synthesized after irradiation [2]. It was originally suggested that the T–T dimers remained in the irradiated strand, but further experimentation using T4 endonuclease V provided proof that T–T dimer exchange did occur between DNA strands [6]. Thus, it appeared that in the absence of excision repair, E. coli cells use a recombination-based mechanism to tolerate UV-induced DNA damage. Despite the initial observation that yeast rad52 cells are defective in PRR [4], further experiments led to a dispute over whether Rad52 was in fact involved in error-free PRR. Resnick et al. [94] examined the level of dimer exchange in postirradiated NER-defective S. cerevisiae cells; the purpose was to

assess whether, as observed in E. coli [6], the DNA strand containing pyrimidine dimers was exchanged with the newly synthesized strand. Since pyrimidine dimer-dependent strand exchange was not observed, it was concluded that a recombination-based mechanism was not involved in PRR. Nevertheless, subsequent studies argue that a recombination mechanism is used for the completion of replication of a damaged DNA template in S. cerevisiae. UV irradiation of NER-deficient diploid rad1/rad1 cells stimulated SCE in a replication-dependent manner [92]. This result was taken to indicate that SCE is a mechanism used by cells to bypass UV-induced damage (and presumably other polymerase-blocking lesions) during DNA synthesis. Two models have been proposed to account for UV-induced SCE (reviewed in [9,95]). In one, the free 3 end of the daughter strand gap invades the sister duplex and a Holliday junction is formed. Branch migration across the damaged portion of the template bypasses the lesion, and resolution of the structure leads to an exchange of genetic information (Fig. 3A). In the second model, the 3 end of the daughter strand gap invades the sister duplex and uses the nascent strand as a template for replicating beyond the lesion on the leading strand template. A second template switch downstream of the lesion restores the normal replication fork. This process is illustrated in Fig. 3B and termed a copy-choice mode of DNA synthesis. Support for the first model came from a study [96] by two-dimensional gel electrophoresis, where Holliday junctions form spontaneously in yeast cells, but only during S phase (at least at the detection level of their assay). It was further demonstrated that the level of Holliday junction formation increased in temperature-sensitive DNA Pol␣ and ␦ mutants held at the restrictive temperature. Thus, Holliday junctions arise in response to arrested DNA replication. The formation of Holliday junctions was dependent upon Rad52, but not on the RecA homologs Rad51, Rad55, or Rad57 [96]. Whether resolution of aberrant replication due to a mutated polymerase is similar to the resolution of damage-induced aberrant replication remains to be answered. Recombination-mediated replication of damaged DNA in mammalian cells might proceed by a copy-choice DNA synthesis mechanism. An early observation [93] pointed to the possibility of strand

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displacement and branch migration creating a template for replicative bypass of a DNA lesion. It was proposed that after the leading strand is blocked, lagging strand synthesis continues for a short distance, replicating beyond the lesion on the leading strand template. The newly synthesized strands are displaced, and the parental and daughter strands anneal (the fork reverses). This creates an alternative template for leading strand DNA synthesis. After synthesis the structure needs to be resolved to restore a normal replication fork [93]. The authors found evidence to support their model. After treatment of cells with MMS and incubation in the presence of BrdU to label newly synthesized DNA, genomic DNA was isolated, sheared, and applied to a neutral CsCl density gradient. Unreplicated parental DNA appears as a light peak, while newly synthesized DNA appears as a peak of intermediate density. There was a third heavy peak of DNA, indicative of conservative DNA synthesis or daughter–daughter annealing. The average length of the DNA molecules in the heavy peak coincide with the length of the short arm of a four-armed structure seen in electron micrographs of the same genomic DNA from MMS-treated cells (short arm = 440–4800 nt). However, this structure could be an experimental artifact.

5. PRR and cell-cycle control

Fig. 3. Two alternative models for error-free PRR via recombinational processes. (A) A strand exchange model, and (B) a template-switching model. Both models propose that progression of leading strand synthesis in the presence of replication-blocking DNA damage (represented by a triangle) requires the association of the two nascent DNA strands, followed by resolution of the intermediary structure via (A) cleavage of the Holliday junction or (B) reverse branch migration.

For PRR to accomplish its function, S phase must accommodate damage-avoidance and TLS modes of DNA replication, both of which are envisioned to extend the time required to complete DNA replication. In E. coli, it is estimated that reactivating the replication fork takes approximately 15–50 min [97–99]. In S. cerevisiae, the S phase progression or intra-S checkpoint slows down the rate of DNA replication in response to DNA damage [100]. This is accomplished at two levels: inhibition of replication fork firing [101,102] and inhibition of replication fork elongation [103]. This checkpoint is fully dependent on RAD53 and MEC1, and partially dependent on RAD9, RAD17, RAD24, MEC3, PRI1, RFA1, and RFC5 [100,103–106]. Two recent studies provide evidence that links PRR to the intra-S checkpoint. UV-induced replication-dependent SCE is likely a consequence of the damage-avoidance PRR activity

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[63]. Not surprisingly, this phenomenon requires RAD52 [92]. The checkpoint genes RAD9 and RAD17 are also required for the maximal induction of SCE after UV irradiation of NER-deficient rad1
cells [63]. It is not known whether this reflects a direct involvement of Rad9 and Rad17 in induced SCE, or whether the effect is due to the role of Rad9 and Rad17 in regulating gene expression in response to DNA damage [107]. A rad1
rad52
mutant synchronized in G1, UV irradiated, and released from arrest, is hypermutable, indicating that at least some of the lesions normally repaired by a SCE event are channeled into a Pol␨-dependent TLS pathway [63]. Interestingly, the checkpoint genes RAD9, RAD17, RAD24, and MEC3 are also required for UV-induced mutagenesis, but not for spontaneous mutagenesis [63]. It was suggested that since Rad24 contains regions of similarity to RF-C subunits and RAD24 displays a genetic interaction with RFC1 [108], perhaps DNA damage during S phase promotes the association of Rad24 with RF-C, facilitating the loading of Pol␨ onto the damaged DNA template [63]. Alternatively, the checkpoint proteins might be required for the transcriptional activation of mutagenesis genes thereby allowing for damage-induced mutagenesis [63] reminiscent of the E. coli SOS response. Observations that deletion of SRS2 rescues rad6 and rad18 cells from killing by a variety of DNA damaging agents [82–84] and that SRS2 channeled replication-blocking lesions from recombination repair to PRR [84] led to a hypothesis that SRS2 functions in a cell-cycle-dependent manner [109]. In haploid yeast cells, SRS2 may inhibit the access of recombination repair to single-stranded gaps during S phase when homologous chromosomes are not available. Srs2 was recently shown to be involved in the intra-S DNA damage checkpoint, and is phosphorylated in response to intra-S DNA damage in a checkpoint-dependent manner [109]. Srs2 phosphorylation is decreased or prevented in mec1, rad53, and dun1
mutants, and by inhibiting the cyclin-dependent kinase Cdk1 [109]. In srs2
mutants arrested in G1 and released into MMS-containing media, Rad53 kinase activity was reduced [109]. This correlated with a premature phosphorylation of the Pol␣–primase B subunit and an accelerated progression through S phase. Interestingly, treatment of srs2
cells with UV or 4NQO did not affect Rad53

activation [109]. SRS2 has a genetic interaction with certain checkpoint genes, as srs2
partially suppresses the MMS-sensitivity of rad17
and rad24
strains [109]. It was hypothesized that since Srs2 phosphorylation correlates with phosphorylation of the lagging strand synthesis machinery [110], perhaps a replication-coupled repair process such as template-switching is promoted by phosphorylated Srs2 to avoid replication-blocking lesions [109]. This might be facilitated by the Srs2 helicase activity. In srs2
cells, a homologous recombination mechanism might act to resolve the stalled replication fork, underlying the hyper-recombinant activity observed in srs2
mutants [85,90,91]. Hence, while the Srs2 helicase is the initiating factor, committing the cell to Rad6-dependent PRR, it is also responsible for providing a liaison between DNA damage tolerance and cell-cycle arrest.

6. Regulation of PRR processes in eukaryotes The relative insensitivity of S. cerevisiae rev3
mutants to UV irradiation as compared to rad6
or rad18
strains (see, for example [64]) suggests that the TLS pathway of DNA Pol␨ is not the preferred pathway for tolerating UV-induced DNA damage encountered during S phase. Alternatively, it could indicate that in the absence of DNA Pol␨, cells can effectively channel the replication-blocking lesions into an error-free tolerance or damage-avoidance pathway. To examine this issue, Baynton et al. [111] designed a plasmid-based in vivo assay to determine the preferred mode of lesion bypass in the presence of a polymerase-blocking N-2-acetylaminofluorene (AAF) adduct. In wild type cells, 92% of the plasmids were replicated using damage-avoidance mechanisms (possibly recombination-based); TLS was used only 8% of the time, and only 3% of that was actually mutagenic. In rev3
cells, no TLS was observed. It should be noted that these results might not extend to other replication-blocking lesions. AAF promotes mutagenesis by frameshifting, not mispairing, and PRR of lesions in a plasmid may differ from that in chromosomal DNA. Nevertheless, these results do suggest that in S. cerevisiae, damage-avoidance is preferred to TLS for tolerating replication-blocking lesions. Similarly, in E. coli cells not induced for the

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SOS response, the same assay yielded less than 1% TLS events. In SOS-induced cells, TLS events are increased to roughly 13% [112]. As expected, deletion of umuDC abolished all TLS in this assay [112]. These results suggest that in E. coli, like in S. cerevisiae, damage-avoidance mechanisms are preferred to TLS for tolerating replication-blocking DNA damage. This issue has also been examined using human cell extracts. Cordeiro-Stone et al. [113] constructed a double-stranded plasmid replicated from the SV40 origin of replication and containing a single T–T dimer placed on the leading strand template of one replication fork. Fork progress was inhibited briefly at the T–T dimer, as observed using two-dimensional gel electrophoresis. T–T dimers were cleaved by T4 endonuclease V in roughly half of the replicated plasmids, as would be expected if the dimer was not removed from the DNA template before or during replication [113]. Xeroderma pigmentosum group A (XPA) cell extracts were competent for replication off of this template, however, XP variant (XPV) extracts were not [113]. In this assay, replication fork bypass of the T–T dimer can occur either by TLS or recombination-mediated template-switching. The assay was modified to enable discrimination between the two possibilities by including a single mismatch opposite or near the T–T dimer on the complementary strand [114]. The sequence of the end products indicated which mechanism was used to complete DNA replication. The results showed that the vast majority of plasmid replication was due to TLS across the T–T dimer [114]. Thus, human and yeast cells appear to differ in their preference for TLS versus damage-avoidance mechanisms, although the above two studies cannot be directly compared because they used a different experimental design and different replication-blocking lesions. More experiments incorporating the same design and replication-blocking lesion are needed before direct comparisons can be made between S. cerevisiae and human damage tolerance mechanisms.

7. PRR in mammalian cells Despite the distinction between prokaryotes and eukaryotes regarding DNA damage tolerance, there appears to be an evolutionary conservation of PRR

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processes within the eukaryotic kingdom. Almost all yeast PRR and mutagenesis pathway genes have mammalian homologs, including hREV1 [115,116], hREV3 [117–119], hREV7 [120], hRAD30/XPV/Pol␩ [121,122], hRAD30B/Pol␫ [123–125], HR6A, HR6B [126,127], hRAD18 [128], hMMS2 [129] and hUBC13 [130]. Some of the above human genes, including HR6A, HR6B [126,127], hMMS2 [129] and hUBC13 (Pastushok and Xiao, unpublished observations), are able to functionally complement the corresponding yeast null mutants with respect to PRR and mutagenesis phenotypes, suggesting that these genes may play a similar role in humans. Furthermore, in a few available studies, suppression of hREV1 [116], hREV3 [118], hRAD18 [128], and hMMS2 [131] gene expression in cultured cells, or deletion of HR6B in transgenic mice [132], resulted in phenotypes characteristic of defective PRR and mutagenesis activities in yeasts, further strengthening the cognate biological roles of these genes in higher eukaryotes.

8. Concluding remarks Among all known DNA repair pathways in eukaryotes, the DNA PRR and mutagenesis pathway is the most complicated and least characterized. Nevertheless, we have witnessed the recent rapid advance in the identification and characterization of non-essential and mutagenic DNA polymerases, and their in vivo roles in TLS are beginning to be elucidated. Advances have also been made in the identification and characterization of genes involved in the error-free mode of PRR. Although very little biochemical characterization has been reported on the PRR proteins, many disparate genetic analyses have yielded clues as to how the process might occur. By integrating the 30 years of genetic information, we believe that a framework has been formed to which future biochemical studies can be directed. We predict that elucidation of the molecular mechanisms of damage-avoidance in eukaryotes will represent the next wave of revolution in the field of DNA repair and mutagenesis. It is well known that mutations in various DNA metabolism/repair genes are associated with an increased likelihood of cancer and other diseases in humans. The XPV gene encoding the human homolog of the yeast Rad30/Pol␩ [121,122] has provided the

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first connection between PRR and human diseases. Thus, the study of eukaryotic PRR and mutagenesis may also increase our awareness of public health.

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