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.     

Cooperation of RAD51 and RAD54 in regression of a model replication fork

DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the 'template-strand switch' mechanism. This mechanism involves replication fork regression into a Holliday junction ('chicken foot structure'), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion.     

Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair.

The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.     

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.     

Cells expressing murine RAD52 splice variants favor sister chromatid repair

The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.     

Rad54, a Swi2/Snf2-like recombinational repair protein,disassembles Rad51:dsDNA filaments

Rad54 protein is a member of the Swi2/Snf2-like family of DNA-dependent/stimulated ATPases that dissociate and remodel protein complexes on dsDNA. Rad54 functions in the recombinational DNA repair (RAD52) pathway. Here we show that Rad54 protein dissociates Rad51 from nucleoprotein filaments formed on dsDNA. Addition of Rad54 protein overcomes inhibition of DNA strand exchange by Rad51 protein bound to substrate dsDNA. Species preference in the Rad51 dissociation and DNA strand exchange assays underlines the importance of specific Rad54-Rad51 protein interactions. Rad51 protein is unable to release dsDNA upon ATP hydrolysis, leaving it stuck on the heteroduplex DNA product after DNA strand exchange. We suggest that Rad54 protein is involved in the turnover of Rad51-dsDNA filaments.     

ATP-dependent chromatin remodeling by the Saccharomyces cerevisiae homologous recombination factor Rdh54

Saccharomyces cerevisiae RDH54 is a key member of the evolutionarily conserved RAD52 epistasis group of genes needed for homologous recombination and DNA double strand break repair. The RDH54-encoded protein possesses a DNA translocase activity and functions together with the Rad51 recombinase in the D-loop reaction. By chromatin immunoprecipitation (ChIP), we show that Rdh54 is recruited, in a manner that is dependent on Rad51 and Rad52, to a site-specific DNA double strand break induced by the HO endonuclease. Because of its relatedness to Swi2/Snf2 chromatin remodelers, we have asked whether highly purified Rdh54 possesses chromatin-remodeling activity. Importantly, our results show that Rdh54 can mobilize a mononucleosome along DNA and render nucleosomal DNA accessible to a restriction enzyme, indicative of a chromatin-remodeling function. Moreover, Rdh54 co-operates with Rad51 in the utilization of naked or chromatinized DNA as template for D-loop formation. We also provide evidence for a strict dependence of the chromatin-remodeling attributes of Rdh54 on its ATPase activity and N-terminal domain. Interestingly, an N-terminal deletion mutant (rdh54Delta102) is unable to promote Rad51-mediated D-loop formation with a chromatinized template, while retaining substantial activity with naked DNA. These features of Rdh54 suggest a role of this protein factor in chromatin rearrangement during DNA recombination and repair.     

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.     

Characterization of RAD51-independent break-induced replication that acts preferentially with short homologous sequences

Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase.     

Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament

RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.     

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.     

Large inverted repeats in the vicinity of a single double-strand break strongly affect repair in yeast diploids lacking Rad51

DNA double-strand breaks (DSBs) are critical lesions that can lead to cell death or chromosomal rearrangements. Rad51 is necessary for most mitotic and meiotic DSB repair events, although a number of RAD51-independent pathways exist. Previously, we described DSB repair in rad51Delta yeast diploids that was stimulated by a DNA region termed "facilitator of break-induced replication" (FBI) located approximately 30kb from the site of an HO-induced DSB. Here, we demonstrate that FBI is a large inverted DNA repeat that channels the repair of DSBs into the single-strand annealing-gross chromosomal rearrangements (SSA-GCR) pathway. Further, analysis of DSB repair in rad54Delta cells allowed us to propose that the SSA-GCR repair pathway is suppressed in the presence of Rad51p. Therefore, an additional role of Rad51 might be to protect eukaryotic genomes from instabilities by preventing chromosomal rearrangements.     

RAD51-independent inverted-repeat recombination by a strand-annealing mechanism

Recombination between inverted repeats is RAD52 dependent, but reduced only modestly in the rad51Δ mutant. RAD59 is required for RAD51-independent inverted-repeat recombination, but no clear mechanism for how recombination occurs in the absence of RAD51 has emerged. Because Rad59 is thought to function as an accessory factor for the single-strand annealing activity of Rad52 one possible mechanism for spontaneous recombination could be by strand annealing between repeats at a stalled replication fork. Here we demonstrate the importance of the Rad52 single-strand annealing activity for generating recombinants by showing suppression of the rad52Δ, rad51Δ rad52Δ and rad52Δ rad59Δ inverted-repeat recombination defects by the rfa1-D228Y mutation. In addition, formation of recombinants in the rad51Δ mutant was sensitive to the distance between the inverted repeats, consistent with a replication-based mechanism. Deletion of RAD5 or RAD18, which are required for error-free post-replication repair, reduced the recombination rate in the rad59Δ mutant, but not in wild type. These data are consistent with RAD51-independent recombinants arising by a faulty template switch mechanism that is distinct from nascent strand template switching.     

Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins.

Rad51p is a eukaryotic homolog of RecA, the central homologous pairing and strand exchange protein in Escherichia coli. Rad54p belongs to the Swi2p/Snf2p family of DNA-stimulated ATPases. Both proteins are also important members of the RAD52 group which controls recombinational DNA damage repair of double-strand breaks and other DNA lesions in Saccharomyces cerevisiae. Here we demonstrate by genetic, molecular and biochemical criteria that Rad51 and Rad54 proteins interact. Strikingly, overexpression of Rad54p can functionally suppress the UV and methyl methanesulfonate sensitivity caused by a deletion of the RAD51 gene. However, no suppression was observed for the defects of rad51 cells in the repair of gamma-ray-induced DNA damage, mating type switching or spontaneous hetero-allelic recombination. This suppression is genetically dependent on the presence of two other members of the recombinational repair group, RAD55 and RAD57. Our data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair. As both proteins are conserved throughout evolution from yeasts to humans, a similar protein-protein interaction may be expected in other organisms.     

Dynamics of some postreplication DNA repair proteins in carcinogen-damaged mammalian cells

Many types of DNA lesions in template strands block DNA replication and lead to a stalling of replication forks. This block can be overcome (bypassed) by special DNA polymerases (for example, DNA polymerase eta, Pol eta) that perform translesion synthesis on damaged template DNA. The phenomenon of completing DNA replication, while DNA lesions remain in the template strands, has been named post-replication repair (PRR). In yeast Saccharomyces cerevisiae, PRR includes mutagenic and error-free pathways under the regulation of the RAD6/RAD18 complex, which induces ubiquitylation of PCNA. In mammalian cells, Pol eta accumulates in replication foci but the mechanism of this accumulation is not known. Pol eta possesses a conserved PCNA binding motif at the C terminal and phosphorylation of this motif might be essential for its interaction with PCNA. We have shown previously that staurosporine, an inhibitor of protein kinases, inhibits PRR in human cells. In this study we examined whether the accumulation of Pol eta in replication foci after DNA damage is dependent on phosphorylation of the PCNA binding motif. We also studied DNA damage-induced phosphorylation of GFP-tagged human Rad18 (hRad18) and its accumulation in replication foci. Our data indicate that (1) Pol eta is not phosphorylated in response to UV irradiation or MMS treatment, but its diffusional mobility is slightly decreased, and (2) hRad18 accumulates in MMS-treated cells, and considerable amount of the protein co-localizes with detergent insoluble PCNA in replication foci; these responses are sensitive to staurosporine. Our data suggest that hRad18 phosphorylation is the staurosporine-sensitive PRR step.     

Homologous DNA pairing by human recombination factors Rad51 and Rad54

Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologous recombination. We show an ability of hRad54 to promote transient separation of the strands in duplex DNA via its ATP hydrolysis-driven DNA supercoiling function. The ATPase, DNA supercoiling, and DNA strand opening activities of hRad54 are greatly stimulated through an interaction with hRad51. Importantly, we demonstrate that hRad51 and hRad54 functionally cooperate in the homologous DNA pairing reaction that forms recombination DNA intermediates. Our results should provide a biochemical model for dissecting the role of hRad51 and hRad54 in recombination reactions in human cells.     

RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.