Takashi Hishida

Homologous recombination is an evolutionally conserved mechanism that promotes genome stability through the faithful repair of double-strand breaks and single-strand gaps in DNA, and the recovery of stalled or collapsed replication forks. Saccharomyces cerevisiae ATP-dependent DNA helicase Srs2 (a member of the highly conserved UvrD family of helicases) has multiple roles in regulating homologous recombination. A mutation (srs2K41A) resulting in a helicase-dead mutant of Srs2 was found to be lethal in diploid, but not in haploid, cells. In diploid cells, Srs2K41A caused the accumulation of inter-homolog joint molecule intermediates, increased the levels of spontaneous Rad52 foci, and induced gross chromosomal rearrangements. Srs2K41A lethality and accumulation of joint molecules were suppressed by inactivating Rad51 or deleting the Rad51-interaction domain of Srs2, whereas phosphorylation and sumoylation of Srs2 and its interaction with sumoylated proliferating cell nuclear antigen (PCNA) were not required for lethality. The structure-specific complex of crossover junction endonucleases Mus81 and Mms4 was also required for viability of diploid, but not haploid, SRS2 deletion mutants (srs2Δ), and diploid srs2Δ mus81Δ mutants accumulated joint molecule intermediates. Our data suggest that Srs2 and Mus81–Mms4 have critical roles in preventing the formation of (or in resolving) toxic inter-homolog joint molecules, which could otherwise interfere with chromosome segregation and lead to genetic instability.

Escherichia coli RecN is an SMC (structural maintenance of chromosomes) family protein that is required for DNA double-strand break (DSB) repair. Previous studies show that GFP-RecN forms nucleoid-associated foci in response to DNA damage, but the mechanism by which RecN is recruited to the nucleoid is unknown. Here, we show that the assembly of GFP-RecN foci on the nucleoid in response to DNA damage involves a functional interaction between RecN and RecA. A novel RecA allele identified in this work, recA(Q300R), is proficient in SOS induction and repair of UV-induced DNA damage, but is deficient in repair of mitomycin C (MMC)-induced DNA damage. Cells carrying recA(Q300R) fail to recruit RecN to DSBs and accumulate fragmented chromosomes after exposure to MMC. The ATPase-deficient RecN(K35A) binds and forms foci at MMC-induced DSBs, but is not released from the MMC-induced DNA lesions, resulting in a defect in homologous recombination-dependent DSB repair. These data suggest that RecN plays a crucial role in homologous recombination-dependent DSB repair and that it is required upstream of RecA-mediated strand exchange.

Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6-RAD18 (an E2-E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2-UBC13 (a UEV-E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13 similar to Ub(n)) and then transfers the chain to RAD6 similar to Ub, forming RAD6 similar to Ub(n+1). The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication.

UV radiation induces two major types of DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidine photoproducts, which are both primarily repaired by nucleotide excision repair (NER). Here, we investigated how chronic low-dose UV (CLUV)-induced mutagenesis occurs in rad14 delta NER-deficient yeast cells, which lack the yeast orthologue of human xeroderma pigmentosum A (XPA). The results show that rad14 delta cells have a marked increase in CLUV-induced mutations, most of which are C -> T transitions in the template strand for transcription. Unexpectedly, many of the CLUV-induced C -> T mutations in rad14 delta cells are dependent on translesion synthesis (TLS) DNA polymerase eta, encoded by RAD30, despite its previously established role in error-free TLS. Furthermore, we demonstrate that deamination of cytosine-containing CPDs contributes to CLUV-induced mutagenesis. Taken together, these results uncover a novel role for Pol eta in the induction of C -> T transitions through deamination of cytosine-containing CPDs in CLUV-exposed NER deficient cells. More generally, our data suggest that Pol eta can act as both an error-free and a mutagenic DNA polymerase, depending on whether the NER pathway is available to efficiently repair damaged templates.

Differential posttranslational modification of proliferating cell nuclear antigen (PCNA) by ubiquitin or SUMO plays an important role in coordinating the processes of DNA replication and DNA damage tolerance. Previously it was shown that the loss of RAD6-dependent error-free postreplication repair (PRR) results in DNA damage checkpoint-mediated G(2) arrest in cells exposed to chronic low-dose UV radiation (CLUV), whereas wild-type and nucleotide excision repair-deficient cells are largely unaffected. In this study, we report that suppression of homologous recombination (HR) in PRR-deficient cells by Srs2 and PCNA sumoylation is required for checkpoint activation and checkpoint maintenance during CLUV irradiation. Cyclin-dependent kinase (CDK1)-dependent phosphorylation of Srs2 did not influence checkpoint-mediated G2 arrest or maintenance in PRR-deficient cells but was critical for HR-dependent checkpoint recovery following release from CLUV exposure. These results indicate that Srs2 plays an important role in checkpoint-mediated reversible G2 arrest in PRR-deficient cells via two separate HR-dependent mechanisms. The first (required to suppress HR during PRR) is regulated by PCNA sumoylation, whereas the second (required for HR-dependent recovery following CLUV exposure) is regulated by CDK1-dependent phosphorylation.

Translesion DNA synthesis (TLS) involves PCNA mono-ubiquitination and TLS DNA polymerases (pols). Recent evidence has shown that the mono-ubiquitination is induced not only by DNA damage but also by other factors that induce stalling of the DNA replication fork. We studied the effect of spontaneous DNA replication errors on PCNA mono-ubiquitination and TLS induction. In the pol1L868F strain, which expressed an error-prone pol alpha, PCNA was spontaneously mono-ubiquitinated. Pol alpha L868F had a rate-limiting step at the extension from mismatched primer termini. Electron microscopic observation showed the accumulation of a single-stranded region at the DNA replication fork in yeast cells. For pol alpha errors, pol zeta participated in a generation of +1 frameshifts. Furthermore, in the pol1L868F strain, UV-induced mutations were lower than in the wild-type and a pol delta mutant strain (pol3-5DV), and deletion of the RAD30 gene (pol eta) suppressed this defect. These data suggest that nucleotide misincorporation by pol alpha induces exposure of single-stranded DNA, PCNA mono-ubiquitination and activates TLS pols.

In nature, organisms are exposed to chronic low- dose ultraviolet light ( CLUV) as opposed to the acute high doses common to laboratory experiments. Analysis of the cellular response to acute high-dose exposure has delineated the importance of direct DNA repair by the nucleotide excision repair pathway(1) and for checkpoint-induced cell cycle arrest in promoting cell survival(2). Here we examine the response of yeast cells to CLUV and identify a key role for the RAD6-RAD18-RAD5 error- free postreplication repair (RAD6 error-free PRR) pathway(3,4) in promoting cell growth and survival. We show that loss of the RAD6 error- free PRR pathway results in DNA-damage-checkpoint- induced G2 arrest in CLUV-exposed cells, whereas wild-type and nucleotide-excision-repair-deficient cells are largely unaffected. Cell cycle arrest in the absence of the RAD6 error- free PRR pathway was not caused by a repair defect or by the accumulation of ultraviolet-induced photoproducts. Notably, we observed increased replication protein A (RPA) and Rad52 - yellow fluorescent protein foci(5) in the CLUV- exposed rad18 Delta cells and demonstrated that Rad52- mediated homologous recombination is required for the viability of the rad18 Delta cells after release from CLUV- induced G2 arrest. These and other data presented suggest that, in response to environmental levels of ultraviolet exposure, the RAD6 error- free PRR pathway promotes replication of damaged templates without the generation of extensive single- stranded DNA regions. Thus, the error- free PRR pathway is specifically important during chronic low- dose ultraviolet exposure to prevent counter- productive DNA checkpoint activation and allow cells to proliferate normally.

The ESC2 gene encodes a protein with two tandem C-terminal SUMO-like domains and is conserved from yeasts to humans. Previous Studies have implicated Esc2 in gene silencing. Here, we explore the functional significance of SUMO-like domains and describe a novel role for Esc2 in promoting genome integrity during DNA replication. This study shows that esc2 Delta cells are modestly sensitive to hydroxy urea (HU) and defective in sister chromatid cohesion and have a reduced life span, and these effects are enhanced by deletion of the RRM3 gene that is a Pif1-like DNA helicase. esc2 Delta rrm3 Delta cells also have a severe growth defect and accumulate DNA damage in late S/G(2). In contrast, esc2 Delta does not enhance the HU sensitivity or sister chromatid cohesion defect in mrc1 Delta cells, but rather partially suppresses both phenotypes. We also show that deletion of both Esc2 SUMO-like domains destabilizes Esc2 protein and functionally inactivates Esc2, but this phenotype is suppressed by an Esc2 variant with an authentic SUMO domain. These results suggest that Esc2 is functionally equivalent to a stable SUMO fusion protein and plays important roles in facilitating DNA replication fork progression and sister chromatid cohesion that would otherwise impede the replication fork in rrm3 Delta cells.

Background: DNA double-strand breaks (DSBs) are induced by exogenous insults such as ionizing radiation and chemical exposure, and they can also arise as a consequence of stalled or collapsed DNA replication forks. Failure to repair DSBs can lead to genomic instability or cell death and cancer in higher eukaryotes. The Schizosaccharomyces pombe fbh1 gene encodes an F-box DNA helicase previously described to play a role in the Rhp51 (an orthologue of S. cerevisiae RAD51)-dependent recombinational repair of DSBs. Fbh1 fused to GFP localizes to discrete nuclear foci following DNA damage. Results: To determine the functional roles of the highly conserved F-box and helicase domains, we have characterized fbh1 mutants carrying specific mutations in these domains. We show that the F-box mutation fbh1-fb disturbs the nuclear localization of Fbh1, conferring an fbh1 null-like phenotype. Moreover, nuclear foci do not form in fbh1-fb cells with DNA damage even if Fbh1-fb is targeted to the nucleus by fusion to a nuclear localization signal sequence. In contrast, the helicase mutation fbh1-hl causes the accumulation of Fbh1 foci irrespective of the presence of DNA damage and confers damage sensitivity greater than that conferred by the null allele. Additional mutation of the F-box alleviates the hypermorphic phenotype of the fbh1-hl mutant. Conclusion: These results suggest that the F-box and DNA helicase domains play indispensable but distinct roles in Fbh1 function. Assembly of the SCFFbh1 complex is required for both the nuclear localization and DNA damage-induced focus formation of Fbh1 and is therefore prerequisite for the Fbh1 recombination function.

Protein degradation in bacteria plays a dynamic and critical role in the cellular response to environmental stimuli such as heat shock and DNA damage and in removing damaged proteins or protein aggregates. Escherichia coli recN is a member of the structural maintenance of chromosomes family and is required for DNA double strand break (DSB) repair. This study shows that RecN protein has a short half-life and its degradation is dependent on the cytoplasmic protease ClpXP and a degradation signal at the C terminus of RecN. In cells with DNA DSBs, green fluorescent protein-RecN localized in discrete foci on nucleoids and formed visible aggregates in the cytoplasm, both of which disappeared rapidly in wild-type cells when DSBs were repaired. In contrast, in Delta clpX cells, RecN aggregates persisted in the cytoplasm after release from DNA damage. Furthermore, analysis of cells experiencing chronic DNA damage revealed that proteolytic removal of RecN aggregates by ClpXP was important for cell viability. These data demonstrate that ClpXP is a critical factor in the cellular clearance of cytoplasmic RecN aggregates from the cell and therefore plays an important role in DNA damage tolerance.

Proliferating cell nuclear antigen (PCNA), a sliding clamp required for processive DNA synthesis, provides attachment sites for various other proteins that function in DNA replication. DNA repair. cell cycle progression and chromatin assembly. It has been shown that differential posttranslational modifications of PCNA by ubiquitin or SUMO play a pivotal role in controlling the choice of pathway for rescuing stalled replication forks. Here, we explored the roles of Mgs1 and PCNA in replication fork rescue. We provide evidence that Mgs1 physically associates with PCNA and that Mgs1 helps suppress the RAD6 DNA damage tolerance pathway in the absence of exogenous DNA damage. We also show that PCNA sumoylation inhibits the growth of mgs1 rad18 double mutants, in which PCNA sumoylation and the Srs2 DNA helicase coordinately prevent RAD52-dependent homologous recombination. The proposed roles for Mgs1. Srs2. and modified PCNA during replication arrest highlight the importance of modulating the RAD6 and RAD52 pathways to avoid genome instability.

The Schizosaccharomyces pombe rad60 gene is essential for cell growth and is involved in repairing DNA double-strand breaks. Rad60 physically interacts with and is functionally related to the structural maintenance of chromosomes 5 and 6 (SMC5/6) protein complex. In this study, we investigated the role of Rad60 in the recovery from the arrest of DNA replication induced by hydroxyurea (HU). rad60-1 mutant cells arrested mitosis normally when treated with HU. Significantly, Rad60 function is not required during HU arrest but is required on release. However, the mutant cells underwent aberrant mitosis accompanied by irregular segregation of chromosomes, and DNA replication was not completed, as revealed by pulsed-field gel electrophoresis. The deletion of rhp51 suppressed the aberrant mitosis of rad60-1 cells and caused mitotic arrest. These results suggest that Rhp51 and Rad60 are required for the restoration of a stalled or collapsed replication fork after release from the arrest of DNA replication by HU. The rad60-1 mutant was proficient in Rhp51 focus formation after release from the HU-induced arrest of DNA replication or DNA-damaging treatment. Furthermore, the lethality of a rad60-1 rqh1 Delta double mutant was suppressed by the deletion of rhp51 or rhp57. These results suggest that Rad60 is required for recombination repair at a step downstream of Rhp51. We propose that Rhp51-dependent DNA structures that cannot activate the mitotic checkpoints accumulate in rad60-1 cells.

RuvB protein forms two hexameric rings that bind to the RuvA tetramer at DNA Holliday junctions. The RuvAB complex utilizes the energy of ATP hydrolysis to promote branch migration of Holliday junctions. The crystal structure of RuvB from Thermus thermophilus (Tth) HB8 showed that each RuvB monomer has three domains (N, M, and C). This study is a structure-function analysis of the three domains of RuvB. The results show that domain N is involved in RuvA-RuvB and RuvB-RuvB subunit interactions, domains N and M are required for ATP hydrolysis and ATP binding-induced hexamer formation, and domain C plays an essential role in DNA binding. The side chain of Arg-318 is essential for DNA binding and may directly interact with DNA. The data also provide evidence that coordinated ATP-dependent interactions between domains N, M, and C play an essential role during formation of the RuvAB Holliday junction ternary complex.

Escherichia coli RecA protein plays a role in DNA homologous recombination, recombination repair, and the rescue of stalled or collapsed replication forks. The mgsA (rarA) gene encodes a highly conserved DNA-dependent ATPase, whose yeast orthologue, MGS1, plays a role in maintaining genomic stability. In this study, we show a functional relationship between mgsA and recA during DNA replication. The mgsA recA double mutant grows more slowly and has lower viability than a recA single mutant, but they are equally sensitive to UV-induced DNA damage. Mutations in mgsA and recA cause lethality in DNA polymerase I deficient cells, and suppress the temperature-dependent growth defect of dnaE486 (Pol III alpha-catalytic subunit). Moreover, recAS25P, a novel recA allele identified in this work, does not complement the slow growth of DeltamgsA DeltarecA cells or the lethality of polA12 DeltarecA, but is proficient in DNA repair, homologous recombination, SOS mutagenesis and SOS induction. These results suggest that RecA and MgsA are functionally redundant in rescuing stalled replication forks, and that the DNA repair and homologous recombination functions of RecA are separated from its function to maintain progression of replication fork.

The RecQ protein family is a highly conserved group of DNA helicases that play roles in maintaining genomic stability. In this study, we present biochemical and genetic evidence that Escherichia coli RecQ processes stalled replication forks and participates in SOS signaling. Cells that carry dnaE486, a mutation in the DNA polymerase III alpha-catalytic subunit, induce an RecA-dependent SOS response and become highly filamented at the semirestrictive temperature (38degreesC). An recQ mutation suppresses the induction of SOS response and the filamentation in the dnaE486 mutant at 38degreesC, causing appearance of a high proportion of anucleate cells. In vitro, RecQ binds and unwinds forked DNA substrates with a gap on the leading strand more efficiently than those with a gap on the lagging strand or Holliday junction DNA. RecQ unwinds the template duplex ahead of the fork, and then the lagging strand is unwound. Consequently, this process generates a single-stranded DNA (ssDNA) gap on the lagging strand adjacent to a replication fork. These results suggest that RecQ functions to generate an initiating signal that can recruit RecA for SOS induction and recombination at stalled replication forks, which are required for the cell cycle checkpoint and resumption of DNA replication.

The Escherichia coli RuvA and RuvB protein complex promotes branch migration of Holliday junctions during recombinational repair and homologous recombination and at stalled replication forks. The RuvB protein belongs to the AAA(+) (ATPase associated with various cellular activities) ATPase family and forms a hexameric ring in an ATP-dependent manner. Studies on the oligomeric AAA+ class ATPases suggest that a conserved arginine residue is located in close proximity to the ATPase site of the adjacent subunit and plays an essential role during ATP hydrolysis. This study presents direct evidence that Arg-174 of RuvB allosterically stimulates the ATPase of the adjacent subunit in a RuvB hexamer. RuvBR174A shows a dominant negative phenotype for DNA repair in vivo and inhibits the branch migration catalyzed by wild-type RuvB. A dominant negative phenotype was also observed with RuvBK68A (Walker A mutation). RuvB K68A-R174A double mutant demonstrates a more severe dominant negative effect than the single mutants RuvB K68A or R174A. Moreover, although RuvB K68A and R174A are totally defective in ATPase activity, ATPase activity is restored when these two mutant proteins are mixed at a 1:1 ratio. These results suggest that each of the two mutants has distinct functional defects and that restoration of the ATPase activity is brought by complementary interaction between the mutant subunits in the heterohexamers. This study demonstrates that R174 plays an intermolecular catalytic role during ATP hydrolysis by RuvB. This role may be a general feature of the oligomeric AAA/AAA(+) ATPases.

Saccharomyces cerevisiae Mgs1 protein, which possesses DNA-dependent ATPase and single strand DNA annealing activities, plays a role in maintaining genomic stability. We found that mgs1 is synthetic lethal with rad6 and exhibits a synergistic growth defect with rad18 and rad5, which are members of the RAD6 epistasis group important for tolerance of DNA damage during DNA replication. The mgs1 mutant is not sensitive to DNA-damaging agents, but the mgs1 rad5 double mutant has increased sensitivity to hydroxyurea and a greatly increased spontaneous mutation rate. Growth defects of mgs1 rad18 double mutants are suppressed by a mutation in SRS2, encoding a DNA helicase, or by overexpression of Rad52. Moreover, mgs1 mutation suppresses the temperature sensitivity of mutants in POL3, encoding DNA polymerase delta. mgs1 also suppresses the growth defect of a pol3 mutant caused by expression of Escherichia coli RuvC, a bacterial Holliday junction resolvase. These findings suggest that Mgs1 is essential for preventing genome instability caused by replication fork arrest in cells deficient in the RAD6 pathway and may modulate replication fork movement catalyzed by yeast polymerase delta.

Background: Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. For junction resolution, RuvC undergoes distinct steps such as dimerization, junction-specific binding and endonucleolytic cleavage. The crystal structure of RuvC has been revealed. Results: To identify functionally important residue, we isolated a large number of mutant ruvC genes created by random mutagenesis and characterized their properties in vivo and in vitro. The mutations which were isolated most frequently were mapped to the four acidic residues constituting the catalytic centre. Amongst the several mutant proteins affected in the dimer interface, only one could not form a dimer. The others were able to form a dimer but were defective in cleavage. F69L and K118R mutant proteins could not cleavage the junction, but they were able to form a dimer and bind the junction DNA. Conclusions: Random mutagenesis highlighted many structurally and functionally important residues of RuvC, most of which highly conserved among RuvC homologues. Dimer formation and also conservation of intact interface interactions between the subunits are important for junction binding and subsequent cleavage. Phe-69 and Lys-118 are critically important for the interactions which lead to junction cleavage.

In Escherichia coli, the products of the ruvA, ruvB and ruvC genes are all involved in the processing of recombination intermediates (Holliday structures) into recombinant molecules. We cloned a 9.4-kb DNA fragment from Pseudomonas aeruginosa PAO1 in a plasmid by functional complementation of the UV sensitivity of an E. coli strain with ruvABC deleted. In P. aeruginosa, the ruv region seemed to form a non-SOS regulated single operon consisting of orf26-ruvC-ruvA-ruvB, while in this region of E. coli, ruvA and ruvB form an SOS-regulated operon, orf26 and ruvC form a non-SOS operon, and these two operons are split by orf23. The deduced amino acid sequences of P. aeruginosa RuvA, RuvB and RuvC proteins were 55, 72 and 55% identical to those of the corresponding E. coli Ruv proteins. The individual ruv genes of P. aeruginosa complemented the corresponding single ruv mutations of E. coli, suggesting that the P. aeruginosa Ruv proteins can interact functionally with their E. coli Ruv partners in forming heterologous complexes. The sequence alignments of the Ruv proteins were extended by incorporation of data about the putative ruv genes obtained from data banks, and the RuvB sequences were conspicuously more conserved than the RuvA and RuvC sequences.