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.     

Opposing effects of ubiquitin conjugation and SUMO modification of PCNA on replicational bypass of DNA lesions in Saccharomyces cerevisiae

The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases zeta (Polzeta) and Poleta and postreplicational repair mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Here we report our studies with a proliferating cell nuclear antigen (PCNA) mutation, pol30-119, which results from a change of the lysine 164 residue to arginine. It has been shown recently that following treatment of yeast cells with DNA-damaging agents, the lysine 164 residue of PCNA becomes monoubiquitinated in a Rad6-Rad18-dependent manner and that subsequently this PCNA residue is polyubiquitinated via a lysine 63-linked ubiquitin chain in an Mms2-Ubc13-, Rad5-dependent manner. PCNA is also modified by SUMO conjugation at the lysine 164 residue. Our genetic studies with the pol30-119 mutation show that in addition to conferring a defect in Polzeta-dependent UV mutagenesis and in Poleta-dependent TLS, this PCNA mutation inhibits postreplicational repair of discontinuities that form in the newly synthesized strand across from UV lesions. In addition, we provide evidence for the activation of the RAD52 recombinational pathway in the pol30-119 mutant and we infer that SUMO conjugation at the lysine 164 residue of PCNA has a role in suppressing the Rad52-dependent postreplicational repair pathway.     

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.     

A role for yeast and human translesion synthesis DNA polymerases in promoting replication through 3-methyl adenine

3-Methyl adenine (3meA), a minor-groove DNA lesion, presents a strong block to synthesis by replicative DNA polymerases (Pols). To elucidate the means by which replication through this DNA lesion is mediated in eukaryotic cells, here we carry out genetic studies in the yeast Saccharomyces cerevisiae treated with the alkylating agent methyl methanesulfonate. From the studies presented here, we infer that replication through the 3meA lesion in yeast cells can be mediated by the action of three Rad6-Rad18-dependent pathways that include translesion synthesis (TLS) by Pol(eta) or -zeta and an Mms2-Ubc13-Rad5-dependent pathway which presumably operates via template switching. We also express human Pols iota and kappa in yeast cells and show that they too can mediate replication through the 3meA lesion in yeast cells, indicating a high degree of evolutionary conservation of the mechanisms that control TLS in yeast and human cells. We discuss these results in the context of previous observations that have been made for the roles of Pols eta, iota, and kappa in promoting replication through the minor-groove N2-dG adducts.     

Requirement of RAD5 and MMS2 for postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

UV lesions in the template strand block the DNA replication machinery. Genetic studies of the yeast Saccharomyces cerevisiae have indicated the requirement of the Rad6-Rad18 complex, which contains ubiquitin-conjugating and DNA-binding activities, in the error-free and mutagenic modes of damage bypass. Here, we examine the contributions of the REV3, RAD30, RAD5, and MMS2 genes, all of which belong to the RAD6 epistasis group, to the postreplication repair of UV-damaged DNA. Discontinuities, which are formed in DNA strands synthesized from UV-damaged templates, are not repaired in the rad5Delta and mms2Delta mutants, thus indicating the requirement of the Rad5 protein and the Mms2-Ubc13 ubiquitin-conjugating enzyme complex in this repair process. Some discontinuities accumulate in the absence of RAD30-encoded DNA polymerase eta (Poleta) but not in the absence of REV3-encoded DNA Polzeta. We concluded that replication through UV lesions in yeast is mediated by at least three separate Rad6-Rad18-dependent pathways, which include mutagenic translesion synthesis by Polzeta, error-free translesion synthesis by Poleta, and postreplication repair of discontinuities by a Rad5-dependent pathway. We suggest that newly synthesized DNA possessing discontinuities is restored to full size by a "copy choice" type of DNA synthesis which requires Rad5, a DNA-dependent ATPase, and also PCNA and Poldelta. The possible roles of the Rad6-Rad18 and the Mms2-Ubc13 enzyme complexes in Rad5-dependent damage bypass are discussed.     

The roles of PCNA SUMOylation, Mms2-Ubc13 and Rad5 in translesion DNA synthesis in Saccharomyces cerevisiae

Mms2, in concert with Ubc13 and Rad5, is responsible for polyubiquitination of replication processivity factor PCNA. This modification activates recombination-like DNA damage-avoidance mechanisms, which function in an error-free manner. Cells deprived of Mms2, Ubc13 or Rad5 exhibit mutator phenotypes as a result of the channelling of premutational DNA lesions to often error-prone translesion DNA synthesis (TLS). Here we show that Siz1-mediated PCNA SUMOylation is required for the stimulation of this TLS, despite the presence of PCNA monoubiquitination. The stimulation of spontaneous mutagenesis by Siz1 in cells carrying rad5 and/or mms2 mutations is connected with the known role of PCNA SUMOylation in the inhibition of Rad52-mediated recombination. However, following UV irradiation, Siz1 is engaged in additional, as yet undefined, mechanisms controlling genetic stability at the replication fork. We also demonstrate that in the absence of PCNA SUMOylation, Mms2-Ubc13 and Rad5 may, independently of each other, function in the stimulation of TLS. Based on this finding and on an analysis of the epistatic relationships between SIZ1, MMS2 and RAD5, with respect to UV sensitivity, we conclude that PCNA SUMOylation is responsible for the functional differences between the Mms2 and Rad5 homologues of Saccharomyces cerevisiae and Schizosaccharomyces pombe.     

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.     

Mms2-Ubc13-dependent and -independent roles of Rad5 ubiquitin ligase in postreplication repair and translesion DNA synthesis in Saccharomyces cerevisiae

The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases eta and zeta and postreplicational repair (PRR) of discontinuities that form in the newly synthesized DNA opposite from DNA lesions, mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Rad5 is an SWI/SNF family ATPase, and additionally, it functions as a ubiquitin ligase in the ubiquitin conjugation reaction. To decipher the roles of these Rad5 activities in lesion bypass, here we examine the effects of mutations in the Rad5 ATPase and ubiquitin ligase domains on the PRR of UV-damaged DNA and on UV-induced mutagenesis. Even though the ATPase-defective mutation confers only a modest degree of UV sensitivity whereas the ubiquitin ligase mutation causes a high degree of UV sensitivity, we find that both of these mutations produce the same high level of PRR defect as that conferred by the highly UV-sensitive rad5Delta mutation. From these studies, we infer a requirement of the Rad5 ATPase and ubiquitin ligase activities in PRR, and based upon the effects of different rad5 mutations on UV mutagenesis, we suggest a role for Rad5 in affecting the efficiency of lesion bypass by the TLS polymerases. In contrast to the role of Rad5 in PRR, however, where its function is coupled with that of Mms2-Ubc13, Rad5 function in TLS would be largely independent of this ubiquitin-conjugating enzyme complex.     

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.     

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。     

PCNA is ubiquitinated by RNF8

The ubiquitination of PCNA is an essential event in the operation of the DNA Damage Tolerance (DDT) pathway that is activated after DNA damage caused by UV or chemical agents during S-phase. This pathway allows the bypass of DNA damage by translesion synthesis that would otherwise cause replication fork stalling. PCNA is mono-ubiquitinated by Rad18-Rad6, and polyubiquitinated by Rad5-Ubc13/Uev1 in the DDT pathway. Mono-and polyubiquitination of PCNA are key processes in the translesion bypass and template switching sub-pathways of the DDT. DNA damage by IR causes DSBs, which trigger the DNA Damage Response (DDR). The ubiquitin ligase RNF8 has a critical role in the assembly of BRCA1 complexes at the DSBs in the DDR. We show that RNF8 readily mono-ubiquitinates PCNA in the presence of UbcH5c, and polyubiquitinates PCNA in the added presence of Ubc13/Uev1a. These reactions are the same as those performed by Rad18-Rad6 and Rad5-Ubc13. RNF8 depletion suppressed both UV and MNNG-stimulated mono-ubiquitination of PCNA, revealing that an RNF8-dependent pathway for PCNA ubiquitination is operative in vivo. These findings provide evidence that RNF8, a key E3 ligase in the DDR, may also play a role in the DDT.     

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.     

Human SHPRH suppresses genomic instability through proliferating cell nuclear antigen polyubiquitination

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63-linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)-induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.     

Translesion DNA synthesis in the context of cancer research

During cell division, replication of the genomic DNA is performed by high-fidelity DNA polymerases but these error-free enzymes can not synthesize across damaged DNA. Specialized DNA polymerases, so called DNA translesion synthesis polymerases (TLS polymerases), can replicate damaged DNA thereby avoiding replication fork breakdown and subsequent chromosomal instability. We focus on the involvement of mammalian TLS polymerases in DNA damage tolerance mechanisms. In detail, we review the discovery of TLS polymerases and describe the molecular features of all the mammalian TLS polymerases identified so far. We give a short overview of the mechanisms that regulate the selectivity and activity of TLS polymerases. In addition, we summarize the current knowledge how different types of DNA damage, relevant either for the induction or treatment of cancer, are bypassed by TLS polymerases. Finally, we elucidate the relevance of TLS polymerases in the context of cancer therapy.     

Regulation of double-stranded DNA gap repair by the RAD6 pathway

The RAD6 pathway allows replication across DNA lesions by either an error-prone or error-free mode. Error-prone replication involves translesion polymerases and requires monoubiquitylation at lysine (K) 164 of PCNA by the Rad6 and Rad18 enzymes. By contrast, the error-free bypass is triggered by modification of PCNA by K63-linked polyubiquitin chains, a reaction that requires in addition to Rad6 and Rad18 the enzymes Rad5 and Ubc13-Mms2. Here, we show that the RAD6 pathway is also critical for controlling repair pathways that act on DNA double-strand breaks. By using gapped plasmids as substrates, we found that repair in wild-type cells proceeds almost exclusively by homology-dependent repair (HDR) using chromosomal DNA as a template, whereas non-homologous end-joining (NHEJ) is suppressed. In contrast, in cells deficient in PCNA polyubiquitylation, plasmid repair occurs largely by NHEJ. Mutant cells that are completely deficient in PCNA ubiquitylation, repair plasmids by HDR similar to wild-type cells. These findings are consistent with a model in which unmodified PCNA supports HDR, whereas PCNA monoubiquitylation diverts repair to NHEJ, which is suppressed by PCNA polyubiquitylation. More generally, our data suggest that the balance between HDR and NHEJ pathways is crucially controlled by genes of the RAD6 pathway through modifications of PCNA.     

Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, postreplication repair (PRR) of UV-damaged DNA occurs by a Rad6-Rad18- and an Mms2-Ubc13-Rad5-dependent pathway or by a Rad52-dependent pathway. The Rad5 DNA helicase activity is specialized for promoting replication fork regression and template switching; previously, we suggested a role for the Rad5-dependent PRR pathway when the lesion is located on the leading strand and a role for the Rad52 pathway when the lesion is located on the lagging strand. In this study, we present evidence for the requirement of Nse1, a subunit of the Smc5-Smc6 complex, in Rad52-dependent PRR, and our genetic analyses suggest a role for the Nse1 and Mms21 E3 ligase activities associated with this complex in this repair mode. We discuss the possible ways by which the Smc5-Smc6 complex, including its associated ubiquitin ligase and SUMO ligase activities, might contribute to the Rad52-dependent nonrecombinational and recombinational modes of PRR.     

Analysis of SHPRH functions in DNA repair and immunoglobulin diversification

During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH in vertebrates. For the latter two enzymes, redundancy with a third E3 ligase and alternative functions have been reported. We have previously shown that the Rad6 pathway is involved in somatic hypermutation of immunoglobulin genes in B lymphocytes. Here, we have used knockout strategies targeting expression of the entire SHPRH protein or functionally significant domains in chicken DT40 cells that do not harbor a HLTF ortholog. We show that SHPRH is apparently redundant with another E3 ligase during DNA damage-induced PCNA modification. SHPRH plays no substantial role in cellular resistance to drugs initiating excision repair and the Rad6 pathway, but is important in survival of topoisomerase II inhibitor treatment. Removal of only the C-terminal RING domain does not interfere with this SHPRH function. SHPRH inactivation does not substantially impact on the overall efficacy of Ig diversification. Redundancy of E3 ligases in the Rad6 pathway may be linked to its different functions in genome maintenance and genetic plasticity.     

Mutations in the ubiquitin binding UBZ motif of DNA polymerase eta do not impair its function in translesion synthesis during replication

Treatment of Saccharomyces cerevisiae cells with DNA-damaging agents elicits lysine 164-linked PCNA monoubiquitination by Rad6-Rad18. Recently, a number of ubiquitin (Ub) binding domains (UBDs) have been identified in translesion synthesis (TLS) DNA polymerases and it has been proposed that the UBD in a TLS polymerase affects its binding to Ub on PCNA and that this binding mode is indispensable for a TLS polymerase to access PCNA at the site of a stalled replication fork. To evaluate the contribution of the binding of UBDs to the Ub moiety on PCNA in TLS, we have examined the effects of mutations in the C2H2 zinc binding motif and in the conserved D570 residue that lies in the alpha-helix portion of the UBZ domain of yeast Poleta. We find that mutations in the C2H2 motif have no perceptible effect on UV sensitivity or UV mutagenesis, whereas a mutation of the D570 residue adversely affects Poleta function. The stimulation of DNA synthesis by Poleta with PCNA or Ub-PCNA was not affected by mutations in the C2H2 motif or the D570 residue. These observations lead us to suggest that the binding of Ub on PCNA via its UBZ domain is not a necessary requirement for the ability of polymerase eta to function in TLS during replication.     

The Mre11-Rad50-Xrs2 Complex Is Required for Yeast DNA Postreplication Repair

Yeast DNA postreplication repair (PRR) bypasses replication-blocking lesions to prevent damage-induced cell death. PRR employs two different mechanisms to bypass damaged DNA, namely translesion synthesis (TLS) and error-free PRR, which are regulated via sequential ubiquitination of proliferating cell nuclear antigen (PCNA). We previously demonstrated that error-free PRR utilizes homologous recombination to facilitate template switching. To our surprise, genes encoding the Mre11-Rad50-Xrs2 (MRX) complex, which are also required for homologous recombination, are epistatic to TLS mutations. Further genetic analyses indicated that two other nucleases involved in double-strand end resection, Sae2 and Exo1, are also variably required for efficient lesion bypass. The involvement of the above genes in TLS and/or error-free PRR could be distinguished by the mutagenesis assay and their differential effects on PCNA ubiquitination. Consistent with the observation that the MRX complex is required for both branches of PRR, the MRX complex was found to physically interact with Rad18 in vivo. In light of the distinct and overlapping activities of the above nucleases in the resection of double-strand breaks, we propose that the interplay between distinct single-strand nucleases dictate the preference between TLS and error-free PRR for lesion bypass.