Role of yeast Rad5 and its human orthologs,HLTF and SHPRH in DNA damage tolerance

In the yeast Saccharomyces cerevisiae, the Rad6–Rad18 DNA damage tolerance pathway constitutes a major defense system against replication fork blocking DNA lesions. The Rad6–Rad18 ubiquitin-conjugating/ligase complex governs error-free and error-prone translesion synthesis by specialized DNA polymerases, as well as an error-free Rad5-dependent postreplicative repair pathway. For facilitating replication through DNA lesions, translesion synthesis polymerases copy directly from the damaged template, while the Rad5-dependent damage tolerance pathway obtains information from the newly synthesized strand of the undamaged sister duplex. Although genetic data demonstrate the importance of the Rad5-dependent pathway in tolerating DNA damages, there has been little understanding of its mechanism. Also, the conservation of the yeast Rad5-dependent pathway in higher order eukaryotic cells remained uncertain for a long time. Here we summarize findings published in recent years regarding the role of Rad5 in promoting error-free replication of damaged DNA, and we also discuss results obtained with its human orthologs, HLTF and SHPRH.     

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.     

Supramolecular complex formation between Rad6 and proteins of the p53 pathway during DNA damage-induced response

The HR6A and -B genes, homologues of the yeast Rad6 gene, encode ubiquitin-conjugating enzymes that are required for postreplication repair of DNA and damage-induced mutagenesis. Using surface plasmon resonance, we show here that HR6 protein (referred as Rad6) physically interacts with p53. Analysis of proteins coimmunoprecipitated with Rad6 antibody from metabolically labeled normal MCF10A human breast epithelial cells not only confirmed Rad6-p53 interactions in vivo but also demonstrated for the first time that exposure of MCF10A cells to cisplatin or adriamycin (ADR) induces recruitment of p14ARF into Rad6-p53 complexes. Further analysis of ADR-induced p53 response showed that stable Rad6-p53-p14ARF complex formation is associated with a parallel increase and decrease in monoubiquitinated and polyubiquitinated p53, respectively, and arrest in G(2)/M phase of the cell cycle. Interestingly, the ADR-induced suppression of p53 polyubiquitination correlated with a corresponding decline in intact Hdm2 protein levels. Treatment of MCF10A cells with MG132, a 26S proteasome inhibitor, effectively stabilized monoubiquitinated p53 and rescued ADR-induced downregulation of Hdm2. These data suggest that ADR-induced degradation of Hdm2 occurs via the ubiquitin-proteasome pathway. Rad6 is present in both the cytoplasmic and nuclear compartments of normal MCF10A cells, although in response to DNA damage it is predominantly found in the nucleus colocalizing with ubiquitinated p53, whereas Hdm2 is undetectable. Consistent with in vivo data, results from in vitro ubiquitination assays show that Rad6 mediates addition of one (mono-) to two (multimono-) ubiquitin molecules on p53 and that inclusion of Mdm2 is essential for its polyubiquitination. The data presented in the present study suggest that Rad6-p53-p14ARF complex formation and p53 ubiquitin modification are important damage-induced responses that perhaps determine the fidelity of DNA postreplication repair.     

<Emphasis Type="Italic">RAD18</Emphasis> gene product of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> controls mutagenesis induced by hydrogen peroxide

Within eukaryotes, tolerance to DNA damage is determined primarily by the repair pathway controlled by the members of the RAD6 epistasis group. Genetic studies on a yeast Saccharomyces cerevisiae model showed that the initial stage of postreplication repair (PRR), i.e., initiation of replication through DNA damage, is controlled by Rad6–Rad18 ubiquitin-conjugating enzyme complex. Mutants of these genes are highly sensitive to various genotoxic agents and reduce the level of induced mutagenesis. In this case, the efficiency of mutagenesis suppression depends on the type of damage. In this study we showed that DNA damage induced by hydrogen peroxide at the same mutagen doses causes significantly more mutations and lethal events in the rad18 mutant cells compared to control wild-type cells.     

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.     

The role of RAD6 in recombinational repair,checkpoints and meiosis via histone modification

The Rad6 ubiquitin-conjugating enzyme in Saccharomyces cerevisiae is known to interact with three separate ubiquitin ligase proteins (Ubr1, Rad18, and Bre1) specific to different targets. The Rad6/Rad18 complex is central to translesion synthesis and the family of DNA transactions known as post-replication repair (PRR). A less well-known aspect of Rad6-mediated DNA repair, however, involves its function with Bre1 in mono-ubiquitinating the histone H2B residue lysine 123. Here, we review how this ubiquitination impacts histone H3 methylation, and how this in turn impacts the DNA damage response. In S. cerevisiae this pathway is required for checkpoint activation in G1, and contributes to DNA repair via the homologous recombination pathway (HRR) in G2 cells. Thus, RAD6 clearly plays a role in HRR in addition to its central role in PRR. We also summarize what is known about related repair pathways in other eukaryotes, including mammals. Recent literature emphasizes the role of methylated histones in S. cerevisiae, Schizosaccharomyces pombe and mammals in attracting the related DNA damage checkpoint proteins Rad9, Crb2 and 53BP1, respectively, to chromatin at the sites of DNA double-strand breaks. However, the specific histone modification pathways involved diverge in these different eukaryotes.     

Domains required for dimerization of yeast Rad6 ubiquitin-conjugating enzyme and Rad18 DNA binding protein.

The RAD6 gene of Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme required for postreplicational repair of UV-damaged DNA and for damage-induced mutagenesis. In addition, Rad6 functions in the N end rule pathway of protein degradation. Rad6 mediates its DNA repair role via its association with Rad18, whose DNA binding activity may target the Rad6-Rad18 complex to damaged sites in DNA. In its role in N end-dependent protein degradation, Rad6 interacts with the UBR1-encoded ubiquitin protein ligase (E3) enzyme. Previous studies have indicated the involvement of N-terminal and C-terminal regions of Rad6 in interactions with Ubr1. Here, we identify the regions of Rad6 and Rad18 that are involved in the dimerization of these two proteins. We show that a region of 40 amino acids towards the C terminus of Rad18 (residues 371 to 410) is sufficient for interaction with Rad6. This region of Rad18 contains a number of nonpolar residues that have been conserved in helix-loop-helix motifs of other proteins. Our studies indicate the requirement for residues 141 to 149 at the C terminus, and suggest the involvement of residues 10 to 22 at the N terminus of Rad6, in the interaction with Rad18. Each of these regions of Rad6 is indicated to form an amphipathic helix.     

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.     

Stability of Rad51 recombinase and persistence of Rad51 DNA repair foci depends on post-translational modifiers,ubiquitin and SUMO

The DNA double-strand breaks are particularly deleterious, especially when an error-free repair pathway is unavailable, enforcing the error-prone recombination pathways to repair the lesion. Cells can resume the cell cycle but at the expense of decreased viability due to genome rearrangements. One of the major players involved in recombinational repair of DNA damage is Rad51 recombinase, a protein responsible for presynaptic complex formation. We previously showed that an increased level of this protein promotes the usage of illegitimate recombination. Here we show that the level of Rad51 is regulated via the ubiquitin-dependent proteolytic pathway. The ubiquitination of Rad51 depends on multiple E3 enzymes, including SUMO-targeted ubiquitin ligases. We also demonstrate that Rad51 can be modified by both ubiquitin and SUMO. Moreover, its modification with ubiquitin may lead to opposite effects: degradation dependent on Rad6, Rad18, Slx8, Dia2, and the anaphase-promoting complex, or stabilization dependent on Rsp5. We also show that post-translational modifications with SUMO and ubiquitin affect Rad51's ability to form and disassemble DNA repair foci, respectively, influencing cell cycle progression and cell viability in genotoxic stress conditions. Our data suggest the existence of a complex E3 ligases network that regulates Rad51 recombinase's turnover, its molecular activity, and access to DNA, limiting it to the proportions optimal for the actual cell cycle stage and growth conditions, e.g., stress. Dysregulation of this network would result in a drop in cell viability due to uncontrolled genome rearrangement in the yeast cells. In mammals would promote the development of genetic diseases and cancer.     

Analysis of the Tolerance to DNA Alkylating Damage in MEC1 and RAD53 Checkpoint Mutants of Saccharomyces cerevisiae

Checkpoint response, tolerance and repair are three major pathways that eukaryotic cells evolved independently to maintain genome stability and integrity. Here, we studied the sensitivity to DNA damage in checkpoint-deficient budding yeast cells and found that checkpoint kinases Mec1 and Rad53 may modulate the balance between error-free and error-prone branches of the tolerance pathway. We have consistently observed that mutation of the RAD53 counterbalances error-free and error-prone branches upon exposure of cells to DNA damage induced either by MMS alkylation or by UV-radiation. We have also found that the potential Mec1/Rad53 balance modulation is independent from Rad6/Rad18-mediated PCNA ubiquitylation, as mec1Δ or rad53Δ mutants show no defects in the modification of the sliding clamp, therefore, we infer that it is likely exerted by acting on TLS polymerases and/or template switching targets.     

The Pol30-K196 residue plays a critical role in budding yeast DNA postreplication repair through interaction with Rad18

PCNA plays critical roles in DNA replication and various DNA repair pathways including DNA damage tolerance (DDT). In budding yeast Saccharomyces cerevisiae, DDT (aka DNA postreplication repair, PRR) is achieved by sequential ubiquitination of PCNA encoded by POL30. Our previous studies revealed that two Arabidopsis PCNA genes were able to complement the essential function of POL30 in budding yeast, but failed to rescue the PRR activity. Here we hypothesize that a certain amino acid variation(s) is responsible for the difference, and identified K196 as a critical residue for the PRR activity. It was found that the pol30-K196V mutation abolishes Rad18 interaction and PRR activity, whereas nearby amino acid substitutions can partially restore Rad18 interaction and PRR activity. Together with the Pol30-Ub fusion data, we believe that we have identified a putative Rad18-binding pocket in Pol30 that is required for PCNA monoubiquitination and PRR.     

Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A

Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.     

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.     

Arabidopsis thaliana UBC13: Implication of Error-free DNA Damage Tolerance and Lys63-linked Polyubiquitylation in Plants

Ubiquitylation is an important biochemical reaction found in all eukaryotic organisms and is involved in a wide range of cellular processes. Conventional ubiquitylation requires the formation of polyubiquitin chains linked through Lys48 of the ubiquitin, which targets specific proteins for degradation. Recently polyubiquitylation through a noncanonical Lys63 chain has been reported, and is required for error-free DNA damage tolerance (or postreplication repair) in yeast. To date, Ubc13 is the only known ubiquitin-conjugating enzyme (Ubc) capable of catalyzing the Lys63-linked polyubiquitylation reaction and this function requires interaction with the Ubc variant Mms2. No information is available on either Lys63-linked ubiquitylation or error-free damage tolerance in plants. We thus cloned and functionally characterized two Arabidopsis thaliana UBC13 genes, AtUBC13A and AtUBC13B. The two genes are highly conserved with respect to chromosomal structure and protein sequence, suggesting that they are derived from a recent gene duplication event. Both AtUbc13 proteins are able to physically interact with yeast or human Mms2, implying that plants also employ the Lys63-linked polyubiquitylation reaction. Furthermore, AtUBC13 genes are able to functionally complement the yeast ubc13 null mutant for spontaneous mutagenesis and sensitivity to DNA damaging agents, suggesting the existence of an error-free DNA damage tolerance pathway in plants. The AtUBC13 genes appear to express ubiquitously and are not induced by various conditions tested.     

Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination.

The human papilloma virus E6-associated protein (E6AP) functions as a ubiquitin protein ligase (E3) in the E6-mediated ubiquitination of p53. E6AP is also an E3 in the absence of E6, but its normal cellular substrates have not yet been identified. Here we report the identification of HHR23A, one of the human homologues of the yeast DNA repair protein Rad23, as an E6-independent target of E6AP. HHR23A binds E6AP and is ubiquitinated in vitro in an E6AP-dependent manner. Ubiquitinated forms of endogenous HHR23A are detectable in mammalian cells. Overexpression of wild-type E6AP in vivo enhances the ubiquitination of HHR23A, whereas a dominant negative E6AP mutant inhibits HHR23A ubiquitination. Although HHR23A is a stable protein in non-synchronized cells, its levels are regulated in a cell cycle-dependent manner, with specific degradation occurring during S phase. The S phase degradation of HHR23A could be blocked in vivo by dominant negative E6AP, providing direct evidence for the involvement of E6AP in the regulation of HHR23A. Consistent with a role of the HHR23 proteins in DNA repair, UV-induced DNA damage inhibited HHR23A degradation. Although the precise role of HHR23 proteins in DNA repair and cell cycle progression remains to be elucidated, our data suggest that E6AP-mediated ubiquitination of HHR23A may have important implications in DNA repair and cell cycle progression.     

Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression

Lesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulate Rad5-dependent postreplicational repair. Here we show that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass.     

Detection of an altered heterochromatin structure in the absence of the nucleotide excision repair protein Rad4 in Saccharomyces cerevisiae

Rad4p is a DNA damage recognition protein essential for global genomic nucleotide excision repair in Saccharomyces cerevisiae. Here, we show that Rad4p binds to the heterochromatic HML locus. In a yeast mutant lacking Rad4p, an increased level of SIR complex binding at the HML locus is accompanied by an altered, more compact heterochromatin structure, as revealed by a topological analysis of chromatin circles released from the locus. In addition, gene silencing at the HML locus is enhanced in the rad4Δ mutant. Importantly, re-expression of Rad4p in the rad4Δ mutant restores the altered heterochromatin structure to a conformation similar to that detected in wild-type cells. These findings reveal a novel role of Rad4p in the regulation of heterochromatin structure and gene silencing.