The yeast Shu complex couples error-free post-replication repair to homologous recombination

DNA post-replication repair (PRR) functions to bypass replication-blocking lesions and prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA. While translesion synthesis has been well characterized, little is known about the molecular events involved in error-free bypass, although it has been assumed that homologous recombination (HR) is required for such a mode of lesion bypass. We undertook a genome-wide synthetic genetic array screen for novel genes involved in error-free PRR and observed evidence of genetic interactions between error-free PRR and HR. Furthermore, this screen identified and assigned four genes, CSM2 , PSY3 , SHU1 and SHU2 , whose products form a stable Shu complex, to the error-free PRR pathway. Previous studies have indicated that the Shu complex is required for efficient HR and that inactivation of any of these genes is able to suppress the severe phenotypes of top3 and sgs1 . We confirmed and further extended some of the reported observations and demonstrated that error-free PRR mutations are also epistatic to sgs1 . Based on the above analyses, we propose a model in which error-free PRR utilizes the Shu complex to recruit HR to facilitate template switching, followed by double-Holliday junction resolution by Sgs1-Top3. This mechanism appears to be conserved throughout eukaryotes.     

The Yeast Shu Complex Utilizes Homologous Recombination Machinery for Error-free Lesion Bypass via Physical Interaction with a Rad51 Paralogue

DNA-damage tolerance (DDT) is defined as a mechanism by which eukaryotic cells resume DNA synthesis to fill the single-stranded DNA gaps left by replication-blocking lesions. Eukaryotic cells employ two different means of DDT, namely translesion DNA synthesis (TLS) and template switching, both of which are coordinately regulated through sequential ubiquitination of PCNA at the K164 residue. In the budding yeast Saccharomyces cerevisiae, the same PCNA-K164 residue can also be sumoylated, which recruits the Srs2 helicase to prevent undesired homologous recombination (HR). While the mediation of TLS by PCNA monoubiquitination has been extensively characterized, the method by which K63-linked PCNA polyubiquitination leads to template switching remains unclear. We recently identified a yeast heterotetrameric Shu complex that couples error-free DDT to HR as a critical step of template switching. Here we report that the Csm2 subunit of Shu physically interacts with Rad55, an accessory protein involved in HR. Rad55 and Rad57 are Rad51 paralogues and form a heterodimer to promote Rad51-ssDNA filament formation by antagonizing Srs2 activity. Although Rad55-Rad57 and Shu function in the same pathway and both act to inhibit Srs2 activity, Shu appears to be dedicated to error-free DDT while the Rad55-Rad57 complex is also involved in double-strand break repair. This study reveals the detailed steps of error-free lesion bypass and also brings to light an intrinsic interplay between error-free DDT and Srs2-mediated inhibition of HR.     

Rad8Rad5/Mms2–Ubc13 ubiquitin ligase complex controls translesion synthesis in fission yeast

Many DNA lesions cause pausing of replication forks at lesion sites; thus, generating gaps in the daughter strands that are filled‐in by post‐replication repair (PRR) pathways. In Saccharomyces cerevisiae, PRR involves translesion synthesis (TLS) mediated by Polη or Polζ, or Rad5‐dependent gap filling through a poorly characterized error‐free mechanism. We have developed an assay to monitor error‐free and mutagenic TLS across single DNA lesions in Schizosaccharomyces pombe. For both main UV photolesions, we have delineated a major error‐free pathway mediated by a distinct combination of TLS polymerases. Surprisingly, these TLS pathways require enzymes needed for poly‐ubiquitination of proliferating cell nuclear antigen (PCNA) as well as those required for mono‐ubiquitination. For pathways that require several TLS polymerases the poly‐ubiquitin chains of PCNA may facilitate their recruitment through specific interactions with their multiple ubiquitin‐binding motifs. These error‐free TLS pathways may at least partially account for the previously described poly‐ubiquitination‐dependent error‐free branch of PRR. This work highlights major differences in the control of lesion tolerance pathways between S. pombe and S. cerevisiae despite the homologous sets of PRR genes these organisms share.     

PCNA modifications for regulation of post-replication repair pathways

Stalled DNA replication forks activate specific DNA repair mechanism called post-replication repair (PRR) pathways that simply bypass DNA damage. The bypassing of DNA damage by PRR prevents prolonged stalling of DNA replication that could result in double strand breaks (DSBs). Proliferating cell nuclear antigen (PCNA) functions to initiate and choose different bypassing pathways of PRR. In yeast, DNA replication forks stalled by DNA damage induces monoubiquitination of PCNA at K164, which is catalyzed by Rad6/Rad18 complex. PCNA monoubiquitination triggers the replacement of replicative polymerase with special translesion synthesis (TLS) polymerases that are able to replicate past DNA lesions. The PCNA interaction motif and/or the ubiquitin binding motif in most TLS polymerases seem to be important for the regulation of TLS. The TLS pathway is usually error-prone because TLS polymerases have low fidelity and no proofreading activity. PCNA can also be further polyubiquitinated by Ubc13/ Mms2/Rad5 complex, which adds an ubiquitin chain onto monoubiquitinated K164 of PCNA. PCNA polyubiquitination directs a different PRR pathway known as error-free damage avoidance, which uses the newly synthesized sister chromatid as a template to bypass DNA damage presumably through template switching mechanism. Mammalian homologues of all of the yeast PRR proteins have been identified, thus PRR is well conserved throughout evolution. Mutations of some PRR genes are associated with a higher risk for cancers in mice and human patients, strongly supporting the importance of PRR as a tumor suppressor pathway.     

受PCNA翻译后修饰调控的DNA损伤耐受机制

为了应对DNA损伤复制阻滞,增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)164位点的赖氨酸残基能够发生一系列的泛素化修饰并介导两种不用的损伤耐受机制,即DNA跨损伤合成(TLS)和无错耐受通路。目前,单泛素化的PCNA介导DNA跨损伤合成通路,而多泛素化的PCNA介导无错耐受通路这一观点已被普遍认可。另外,PCNA的164位点还能被泛素类似物小蛋白(SUMO)修饰,从而抑制DNA双链断裂重组。总结PCNA的翻译后修饰及其在DNA损伤应答过程中的作用机制,有助于我们了解PCNA在DNA损伤耐受机制中的中心作用。重点总结PCNA的翻译后修饰如何调控真核生物DNA损伤应答的不同途径。     

Requirement of Rad5 for DNA polymerase zeta-dependent translesion synthesis in Saccharomyces cerevisiae

In yeast, Rad6-Rad18-dependent lesion bypass involves translesion synthesis (TLS) by DNA polymerases eta or zeta or Rad5-dependent postreplication repair (PRR) in which error-free replication through the DNA lesion occurs by template switching. Rad5 functions in PRR via its two distinct activities-a ubiquitin ligase that promotes Mms2-Ubc13-mediated K63-linked polyubiquitination of PCNA at its lysine 164 residue and a DNA helicase that is specialized for replication fork regression. Both these activities are important for Rad5's ability to function in PRR. Here we provide evidence for the requirement of Rad5 in TLS mediated by Polzeta. Using duplex plasmids carrying different site-specific DNA lesions-an abasic site, a cis-syn TT dimer, a (6-4) TT photoproduct, or a G-AAF adduct-we show that Rad5 is needed for Polzeta-dependent TLS. Rad5 action in this role is likely to be structural, since neither the inactivation of its ubiquitin ligase activity nor the inactivation of its helicase activity impairs its role in TLS.     

RAD18 lives a double life: Its implication in DNA double-strand break repair

Maintenance of genome stability depends on efficient and accurate repair of DNA lesions. Failure to properly repair damaged DNA can cause cell death, mutations and chromosomal instability, which eventually lead to tumorigenesis. The E3 ligase RAD18 is well-known for its function in DNA damage bypass and post-replication repair (PRR) in yeast and vertebrates via its ability to facilitate PCNA mono-ubiquitination at stalled replication forks. However, emerging evidence has also indicated that RAD18 plays an important role in homologous recombination (HR) in mammalian cells, which is an error-free DNA repair pathway that mediates the repair of double-strand breaks (DSBs). Here, we review how RAD18 carries out these distinct functions in response to different types of DNA lesions.     

The Rad5 helicase activity is dispensable for error-free DNA post-replication repair

DNA post-replication repair (PRR) functions to bypass replication-blocking lesions and is subdivided into two parallel pathways: error-prone translesion DNA synthesis and error-free PRR. While both pathways are dependent on the ubiquitination of PCNA, error-free PRR utilizes noncanonical K63-linked polyubiquitinated PCNA to signal lesion bypass through template switch, a process thought to be dependent on Mms2-Ubc13 and a RING finger motif of the Rad5 ubiquitin ligase. Previous in vitro studies demonstrated the ability of Rad5 to promote replication fork regression, a function dependent on its helicase activity. To investigate the genetic and mechanistic relationship between fork regression in vitro and template switch in vivo, we created and characterized site-specific mutations defective in the Rad5 RING or helicase activity. Our results indicate that both the Rad5 ubiquitin ligase and the helicase activities are exclusively involved in the same error-free PRR pathway. Surprisingly, the Rad5 helicase mutation abolishes its physical interaction with Ubc13 and the K63-linked PCNA polyubiquitin chain assembly. Indeed, physical fusions of Rad5 with Ubc13 bypass the requirement for either the helicase or the RING finger domain. Since the helicase domain overlaps with the SWI/SNF chromatin-remodelling domain, our findings suggest a structural role of this domain and that the Rad5 helicase activity is dispensable for error-free lesion bypass.     

Altered translesion synthesis in E. coli Pol V mutants selected for increased recombination inhibition

Replication of damaged DNA, also termed as translesion synthesis (TLS), involves specialized DNA polymerases that bypass DNA lesions. In Escherichia coli, although TLS can involve one or a combination of DNA polymerases depending on the nature of the lesion, it generally requires the Pol V DNA polymerase (formed by two SOS proteins, UmuD' and UmuC) and the RecA protein. In addition to being an essential component of translesion DNA synthesis, Pol V is also an antagonist of RecA-mediated recombination. We have recently isolated umuD' and umuC mutants on the basis of their increased capacity to inhibit homologous recombination. Despite the capacity of these mutants to form a Pol V complex and to interact with the RecA polymer, most of them exhibit a defect in TLS. Here, we further characterize the TLS activity of these Pol V mutants in vivo by measuring the extent of error-free and mutagenic bypass at a single (6-4)TT lesion located in double stranded plasmid DNA. TLS is markedly decreased in most Pol V mutants that we analyzed (8/9) with the exception of one UmuC mutant (F287L) that exhibits wild-type bypass activity. Somewhat unexpectedly, Pol V mutants that are partially deficient in TLS are more severely affected in mutagenic bypass compared to error-free synthesis. The defect in bypass activity of the Pol V mutant polymerases is discussed in light of the location of the respective mutations in the 3D structure of UmuD' and the DinB/UmuC homologous protein Dpo4 of Sulfolobus solfataricus.     

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.     

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

In addition to well-defined DNA repair pathways, all living organisms have evolved mechanisms to avoid cell death caused by replication fork collapse at a site where replication is blocked due to disruptive covalent modifications of DNA. The term DNA damage tolerance （DDT） has been employed loosely to include a collection of mechanisms by which cells survive replication-blocking lesions with or without associated genomic instability. Recent genetic analyses indicate that DDT in eukaryotes, from yeast to human, consists of two parallel pathways with one being error-free and another highly mutagenic. Interestingly, in budding yeast, these two pathways are mediated by sequential modifications of the proliferating cell nuclear antigen （PCNA） by two ubiquitination complexes Rad6-Rad18 and Mms2-Ubc13-Rad5. Damage-induced monoubiquitination of PCNA by Rad6-Rad18 promotes translesion synthesis （TLS） with increased mutagenesis, while subsequent polyubiquitination of PCNA at the same K164 residue by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Data obtained from recent studies suggest that the above mechanisms are conserved in higher eukaryotes. In particular, mammals contain multiple specialized TLS polymerases. Defects in one of the TLS polymerases have been linked to genomic instability and cancer.     

PCNA ubiquitination and REV1 define temporally distinct mechanisms for controlling translesion synthesis in the avian cell line DT40

Translesion synthesis (TLS) is a potentially mutagenic method of bypassing DNA damage encountered during replication that requires the recruitment of specialized DNA polymerases to stalled replication forks or postreplicative gaps. Current models suggest that TLS is activated by monoubiquitination of the DNA sliding clamp PCNA. However, in higher organisms, fully effective TLS also requires a noncatalytic function of the Y family polymerase REV1. Using the genetically tractable chicken cell line DT40, we show that TLS at stalled replication forks requires that both the translesion polymerase-interaction domain and ubiquitin-binding domain in the C terminus of REV1 are intact. Surprisingly, however, PCNA ubiquitination is not required to maintain normal fork progression on damaged DNA. Conversely, PCNA ubiquitination is essential for filling postreplicative gaps. Thus, PCNA ubiquitination and REV1 play distinct roles in the coordination of DNA damage bypass that are temporally separated relative to replication fork arrest.     

PCNA-Ub polyubiquitination inhibits cell proliferation and induces cell-cycle checkpoints

In response to replication-blocking lesions, proliferating cell nuclear antigen (PCNA) can be sequentially ubiquitinated at the K164 residue leading to 2 modes of DNA-damage tolerance, namely translesion DNA synthesis (TLS) and error-free lesion bypass. Ectopic expression of PCNA fused with ubiquitin (Ub) lacking the 2 C-terminal Gly residues resembles PCNA monoubiquitination-mediated TLS. However, if the fused Ub contains C-terminal Gly residues, it is further polyubiquitinated and inhibits cell proliferation. Unexpectedly, the polyubiquitination chain does not require any surface Lys residues and is likely to be head-to-tail linked. Such PCNA polyubiquitination interferes with replication, arrests cells at the S-phase and activates the p53 checkpoint pathway. The above cell-cycle arrest is reversible in an ATR-dependent manner, as simultaneous inhibition of ATR, but not ATM, induces apoptosis. Since ectopic expression of PCNA-Ub also induces double-strand breaks that colocalize with single-stranded DNA, we infer that this non-canonical PCNA poly-Ub chain serves as a signal to activate ATR checkpoint and recruit double-strand-break repair apparatus.     

A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

Replication forks stall at DNA lesions or as a result of an unfavorable replicative environment. These fork stalling events have been associated with recombination and gross chromosomal rearrangements. Recombination and fork bypass pathways are the mechanisms accountable for restart of stalled forks. An important lesion bypass mechanism is the highly conserved post-replication repair (PRR) pathway that is composed of error-prone translesion and error-free bypass branches. EXO1 codes for a Rad2p family member nuclease that has been implicated in a multitude of eukaryotic DNA metabolic pathways that include DNA repair, recombination, replication, and telomere integrity. In this report, we show EXO1 functions in the MMS2 error-free branch of the PRR pathway independent of the role of EXO1 in DNA mismatch repair (MMR). Consistent with the idea that EXO1 functions independently in two separate pathways, we defined a domain of Exo1p required for PRR distinct from those required for interaction with MMR proteins. We then generated a point mutant exo1 allele that was defective for the function of Exo1p in MMR due to disrupted interaction with Mlh1p, but still functional for PRR. Lastly, by using a compound exo1 mutant that was defective for interaction with Mlh1p and deficient for nuclease activity, we provide further evidence that Exo1p plays both structural and catalytic roles during MMR.     

RAD5a and REV3 function in two alternative pathways of DNA-damage tolerance in Arabidopsis

DNA-damage tolerance (DDT) in yeast is composed of two parallel pathways and mediated by sequential ubiquitinations of PCNA. While monoubiquitination of PCNA promotes translesion synthesis (TLS) that is dependent on polymerase ζ consisted of a catalytic subunit Rev3 and a regulatory subunit Rev7, polyubiquitination of PCNA by Mms2-Ubc13-Rad5 promotes error-free lesion bypass. Inactivation of these two pathways results in a synergistic effect on DNA-damage responses; however, this two-branch DDT model has not been reported in any multicellular organisms. In order to examine whether Arabidopsis thaliana possesses a two-branch DDT system, we created rad5a rev3 double mutant plant lines and compared them with the corresponding single mutants. Arabidopsis rad5a and rev3 mutations are indeed synergistic with respect to root growth inhibition induced by replication-blocking lesions, suggesting that AtRAD5a and AtREV3 are required for error-free and TLS branches of DDT, respectively. Unexpectedly this study reveals three modes of genetic interactions in response to different types of DNA damage, implying that plant RAD5 and REV3 are also involved in DNA damage responses independent of DDT. By comparing with yeast cells, it is apparent that plant TLS is a more frequently utilized means of lesion bypass than error-free DDT in plants.     

Eukaryotic stress–induced mutagenesis is limited by a local control of translesion synthesis

The DNA damage response (DDR) preserves the genetic integrity of the cell by sensing and repairing damages after a genotoxic stress. Translesion Synthesis (TLS), an error-prone DNA damage tolerance pathway, is controlled by PCNA ubiquitination. In this work, we raise the question whether TLS is controlled locally or globally. Using a recently developed method that allows to follow the bypass of a single lesion inserted into the yeast genome, we show that (i) TLS is controlled locally at each individual lesion by PCNA ubiquitination, (ii) a single lesion is enough to induce PCNA ubiquitination and (iii) PCNA ubiquitination is imperative for TLS to occur. More importantly, we show that the activation of the DDR that follows a genotoxic stress does not increase TLS at individual lesions. We conclude that unlike the SOS response in bacteria, the eukaryotic DDR does not promote TLS and mutagenesis.     

DNA repair defects channel interstrand DNA cross-links into alternate recombinational and error-prone repair pathways

The repair of psoralen interstrand cross-links in the yeast Saccharomyces cerevisiae involves the DNA repair groups nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (PRR). In repair-proficient yeast cells cross-links induce double-strand breaks, in an NER-dependent process; the double-strand breaks are then repaired by HR. An alternate error-prone repair pathway generates mutations at cross-link sites. We have characterized the repair of plasmid molecules carrying a single psoralen cross-link, psoralen monoadduct, or double-strand break in yeast cells with deficiencies in NER, HR, or PRR genes, measuring the repair efficiencies and the levels of gene conversions, crossing over, and mutations. Strains with deficiencies in the NER genes RAD1, RAD3, RAD4, and RAD10 had low levels of cross-link-induced recombination but higher mutation frequencies than repair-proficient cells. Deletion of the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57 also decreased induced recombination and increased mutation frequencies above those of NER-deficient yeast. Strains lacking the PRR genes RAD5, RAD6, and RAD18 did not have any cross-link-induced mutations but showed increased levels of recombination; rad5 and rad6 cells also had altered patterns of cross-link-induced gene conversion in comparison with repair-proficient yeast. Our observations suggest that psoralen cross-links can be repaired by three pathways: an error-free recombinational pathway requiring NER and HR and two PRR-dependent error-prone pathways, one NER-dependent and one NER-independent.     

The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.     

CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1

Topoisomerases class II (topoII) cleave and re-ligate the DNA double helix to allow the passage of an intact DNA strand through it. Chemotherapeutic drugs such as etoposide target topoII, interfere with the normal enzymatic cleavage/re-ligation reaction and create a DNA double-strand break (DSB) with the enzyme covalently bound to the 5'-end of the DNA. Such DSBs are repaired by one of the two major DSB repair pathways, non-homologous end-joining (NHEJ) or homologous recombination. However, prior to repair, the covalently bound topoII needs to be removed from the DNA end, a process requiring the MRX complex and ctp1 in fission yeast. CtIP, the mammalian ortholog of ctp1, is known to promote homologous recombination by resecting DSB ends. Here, we show that human cells arrested in G0/G1 repair etoposide-induced DSBs by NHEJ and, surprisingly, require the MRN complex (the ortholog of MRX) and CtIP. CtIP's function for repairing etoposide-induced DSBs by NHEJ in G0/G1 requires the Thr-847 but not the Ser-327 phosphorylation site, both of which are needed for resection during HR. This finding establishes that CtIP promotes NHEJ of etoposide-induced DSBs during G0/G1 phase with an end-processing function that is distinct to its resection function.