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Stephen C. Kowalczykowski - One of the best experts on this subject based on the ideXlab platform.
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Rad51 Protein Controls RAD52-mediated DNA Annealing
The Journal of biological chemistry, 2008Co-Authors: Noriko Kantake, Tomohiko Sugiyama, Stephen C. KowalczykowskiAbstract:In Saccharomyces cerevisiae, RAD52 protein plays an essential role in the repair of DNA double-stranded breaks (DSBs). RAD52 and its orthologs possess the unique capacity to anneal single-stranded DNA (ssDNA) complexed with its cognate ssDNA-binding protein, RPA. This annealing activity is used in multiple mechanisms of DSB repair: single-stranded annealing, synthesis-dependent strand annealing, and cross-over formation. Here we report that the S. cerevisiae DNA strand exchange protein, Rad51, prevents RAD52-mediated annealing of complementary ssDNA. Efficient inhibition is ATP-dependent and involves a specific interaction between Rad51 and RAD52. Free Rad51 can limit DNA annealing by RAD52, but the Rad51 nucleoprotein filament is even more effective. We also discovered that the budding yeast RAD52 paralog, Rad59 protein, partially restores RAD52-dependent DNA annealing in the presence of Rad51, suggesting that RAD52 and Rad59 function coordinately to enhance recombinational DNA repair either by directing the processed DSBs to repair by DNA strand annealing or by promoting second end capture to form a double Holliday junction. This regulation of RAD52-mediated annealing suggests a control function for Rad51 in deciding the recombination path taken for a processed DNA break; the ssDNA can be directed to either Rad51-mediated DNA strand invasion or to RAD52-mediated DNA annealing. This channeling determines the nature of the subsequent repair process and is consistent with the observed competition between these pathways in vivo.
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RAD52 mediated dna annealing after rad51 mediated dna strand exchange promotes second ssdna capture
The EMBO Journal, 2006Co-Authors: Tomohiko Sugiyama, Noriko Kantake, Stephen C. KowalczykowskiAbstract:Rad51, RAD52, and RPA play central roles in homologous DNA recombination. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. RAD52 has two distinct activities: to recruit Rad51 onto single-strand (ss)DNA that is complexed with the ssDNA-binding protein, RPA, and to anneal complementary ssDNA complexed with RPA. Here, we report that RAD52 promotes annealing of the ssDNA strand that is displaced by DNA strand exchange by Rad51 and RPA, to a second ssDNA strand. An RPA that is recombination-deficient (RPA(rfa1-t11)) failed to support annealing, explaining its in vivo phenotype. Escherichia coli RecO and SSB proteins, which are functional homologues of RAD52 and RPA, also facilitated the same reaction, demonstrating its conserved nature. We also demonstrate that the two activities of RAD52, recruiting Rad51 and annealing DNA, are coordinated in DNA strand exchange and second ssDNA capture.
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The DNA binding preference of RAD52 and RAD59 proteins: implications for RAD52 and RAD59 protein function in homologous recombination.
The Journal of biological chemistry, 2006Co-Authors: Joseph S. Siino, Tomohiko Sugiyama, Stephen C. KowalczykowskiAbstract:We examined the double-stranded DNA (dsDNA) binding preference of the Saccharomyces cerevisiae RAD52 protein and its homologue, the Rad59 protein. In nuclease protection assays both proteins protected an internal sequence and the dsDNA ends equally well. Similarly, using electrophoretic mobility shift assays, we found the affinity of both RAD52 and Rad59 proteins for DNA ends to be comparable with their affinity for internal sequences. The protein-DNA complexes were also directly visualized using atomic force microscopy. Both proteins formed discrete complexes, which were primarily found (90–94%) at internal dsDNA sites. We also measured the DNA end binding behavior of human RAD52 protein and found a slight preference for dsDNA ends. Thus, these proteins have no strong preference for dsDNA ends over internal sites, which is inconsistent with their function at a step of dsDNA break repair that precedes DNA processing. Therefore, we conclude that S. cerevisiae RAD52 and Rad59 proteins and their eukaryotic counterparts function by binding to single-stranded DNA formed as intermediates of recombination rather than by binding to the unprocessed DNA double-strand break.
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DNA Annealing Mediated by RAD52 and Rad59 Proteins
The Journal of biological chemistry, 2006Co-Authors: Tomohiko Sugiyama, Stephen C. KowalczykowskiAbstract:In the budding yeast Saccharomyces cerevisiae, the RAD52 gene is essential for all homologous recombination events and its homologue, the RAD59 gene, is important for those that occur independently of RAD51. Both RAD52 and Rad59 proteins can anneal complementary single-stranded (ss) DNA. We quantitatively examined the ssDNA annealing activity of RAD52 and Rad59 proteins and found significant differences in their biochemical properties. First, and most importantly, they differ in their ability to anneal ssDNA that is complexed with replication protein A (RPA). RAD52 can anneal an RPA-ssDNA complex, but Rad59 cannot. Second, Rad59-promoted DNA annealing follows first-order reaction kinetics, whereas RAD52-promoted annealing follows second-order reaction kinetics. Last, Rad59 enhances RAD52-mediated DNA annealing at increased NaCl concentrations, both in the absence and presence of RPA. These results suggest that Rad59 performs different functions in the recombination process, and should be more accurately viewed as a RAD52 paralogue.
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rad54 protein possesses chromatin remodeling activity stimulated by the rad51 ssdna nucleoprotein filament
Nature Structural & Molecular Biology, 2003Co-Authors: Andrei A Alexeev, Alexander V Mazin, Stephen C. KowalczykowskiAbstract:In Saccharomyces cerevisiae, the Rad54 protein participates in the recombinational repair of double-strand DNA breaks together with the Rad51, RAD52, Rad55 and Rad57 proteins. In vitro, Rad54 interacts with Rad51 and stimulates DNA strand exchange promoted by Rad51 protein. Rad54 is a SWI2/SNF2-related protein that possesses double-stranded DNA-dependent ATPase activity and changes DNA topology in an ATP hydrolysis-dependent manner. Here we show that Rad54 catalyzes bidirectional nucleosome redistribution by sliding nucleosomes along DNA. Nucleosome redistribution is greatly stimulated by the Rad51 nucleoprotein filament but does not require the presence of homologous single-stranded DNA within the filament. On the basis of these data, we propose that Rad54 facilitates chromatin remodeling and, perhaps more generally, protein clearing at the homology search step of genetic recombination.
Patrick Sung - One of the best experts on this subject based on the ideXlab platform.
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human RAD52 interactions with replication protein a and the rad51 presynaptic complex
Journal of Biological Chemistry, 2017Co-Authors: Chu Jian, Youngho Kwon, Patrick Sung, Eric C GreeneAbstract:RAD52 is a highly conserved protein involved in the repair of DNA damage. Human RAD52 has been shown to mediate single-stranded DNA (ssDNA) and is synthetic lethal with mutations in other key recombination proteins. For this study, we used single-molecule imaging and ssDNA curtains to examine the binding interactions of human RAD52 with replication protein A (RPA)-coated ssDNA, and we monitored the fate of RAD52 during assembly of the presynaptic complex. We show that RAD52 binds tightly to the RPA-ssDNA complex and imparts an inhibitory effect on RPA turnover. We also found that during presynaptic complex assembly, most of the RPA and RAD52 was displaced from the ssDNA, but some RAD52-RPA-ssDNA complexes persisted as interspersed clusters surrounded by RAD51 filaments. Once assembled, the presence of RAD51 restricted formation of new RAD52-binding events, but additional RAD52 could bind once RAD51 dissociated from the ssDNA. Together, these results provide new insights into the behavior and dynamics of human RAD52 during presynaptic complex assembly and disassembly.
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promotion of presynaptic filament assembly by the ensemble of s cerevisiae rad51 paralogues with RAD52
Nature Communications, 2015Co-Authors: William A Gaines, Patrick Sung, Stephen K Godin, Faiz Kabbinavar, Timsi Rao, Andrew P Vandemark, Kara A BernsteinAbstract:The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and RAD52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55–Rad57 and RAD52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2–F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55–Rad57, the Shu complex and RAD52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.
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role of the RAD52 amino terminal dna binding activity in dna strand capture in homologous recombination
Journal of Biological Chemistry, 2009Co-Authors: Idina Shi, Rodney Rothstein, Swee Chuang Lim Hallwyl, Uffe Hasbro Mortensen, Changhyun Seong, Patrick SungAbstract:Saccharomyces cerevisiae RAD52 protein promotes homologous recombination by nucleating the Rad51 recombinase onto replication protein A-coated single-stranded DNA strands and also by directly annealing such strands. We show that the purified RAD52-R70A mutant protein, with a compromised amino-terminal DNA binding domain, is capable of Rad51 delivery to DNA but is deficient in DNA annealing. Results from chromatin immunoprecipitation experiments find that RAD52-R70A associates with DNA double-strand breaks and promotes recruitment of Rad51 as efficiently as wild-type RAD52. Analysis of gene conversion intermediates reveals that RAD52-R70A cells can mediate DNA strand invasion but are unable to complete the recombination event. These results provide evidence that DNA binding by the evolutionarily conserved amino terminus of RAD52 is needed for the capture of the second DNA end during homologous recombination.
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regulation of rad51 recombinase presynaptic filament assembly via interactions with the RAD52 mediator and the srs2 anti recombinase
Journal of Biological Chemistry, 2009Co-Authors: Changhyun Seong, Youngho Kwon, Patrick Sung, Lumir Krejci, Sierra A ColavitoAbstract:Homologous recombination represents an important means for the error-free elimination of DNA double-strand breaks and other deleterious DNA lesions from chromosomes. The Rad51 recombinase, a member of the RAD52 group of recombination proteins, catalyzes the homologous recombination reaction in the context of a helical protein polymer assembled on single-stranded DNA (ssDNA) that is derived from the nucleolytic processing of a primary lesion. The assembly of the Rad51-ssDNA nucleoprotein filament, often referred to as the presynaptic filament, is prone to interference by the single-strand DNA-binding factor replication protein A (RPA). The Saccharomyces cerevisiae RAD52 protein facilitates presynaptic filament assembly by helping to mediate the displacement of RPA from ssDNA. On the other hand, disruption of the presynaptic filament by the Srs2 helicase leads to a net exchange of Rad51 for RPA. To understand the significance of protein-protein interactions in the control of RAD52- or Srs2-mediated presynaptic filament assembly or disassembly, we have examined two rad51 mutants, rad51 Y388H and rad51 G393D, that are simultaneously ablated for RAD52 and Srs2 interactions and one, rad51 A320V, that is differentially inactivated for RAD52 binding for their biochemical properties and also for functional interactions with RAD52 or Srs2. We show that these mutant rad51 proteins are impervious to the mediator activity of RAD52 or the disruptive function of Srs2 in concordance with their protein interaction defects. Our results thus provide insights into the functional significance of the Rad51-RAD52 and Rad51-Srs2 complexes in the control of presynaptic filament assembly and disassembly. Moreover, our biochemical studies have helped identify A320V as a separation-of-function mutation in Rad51 with regards to a differential ablation of RAD52 interaction.
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interaction with rpa is necessary for RAD52 repair center formation and for its mediator activity
Journal of Biological Chemistry, 2008Co-Authors: Iben Plate, Patrick Sung, Swee Chuang Lim Hallwyl, Idina Shi, Lumir Krejci, Christian Muller, Line Albertsen, Uffe Hasbro MortensenAbstract:Homologous recombination (HR) is a major DNA repair pathway and therefore essential for maintaining the integrity of the genome. HR is catalyzed by proteins encoded by genes of the RAD52 epistasis group, including the recombinase Rad51 and its mediator RAD52. HR proteins fused with green fluorescent protein form foci at damaged DNA reflecting the assembly of repair centers that harbor a high concentration of repair proteins. RAD52 mediates the recruitment of Rad51 and other HR proteins to DNA damage. To understand the mechanism for the assembly of RAD52-dependent DNA repair centers, we used a mutational strategy to identify a RAD52 domain essential for its recruitment to DNA repair foci. We present evidence to implicate an acidic domain in RAD52 in DNA repair focus formation. Mutations in this domain confer marked DNA damage sensitivity and recombination deficiency. Importantly, these RAD52 mutants are specifically compromised for interaction with the single-stranded DNA-binding factor RPA. Based on these findings, we propose a model where RAD52 displaces RPA from single-stranded DNA using the acidic domain as a molecular lever.
Akira Shinohara - One of the best experts on this subject based on the ideXlab platform.
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RAD52 Promotes Postinvasion Steps of Meiotic Double-Strand-Break Repair
Molecular cell, 2008Co-Authors: Jessica P. Lao, Miki Shinohara, Akira Shinohara, Neil HunterAbstract:During DNA double-strand-break (DSB) repair by recombination, the broken chromosome uses a homologous chromosome as a repair template. Early steps of recombination are well characterized: DSB ends assemble filaments of RecA-family proteins that catalyze homologous pairing and strand-invasion reactions. By contrast, the postinvasion steps of recombination are poorly characterized. RAD52 plays an essential role during early steps of recombination by mediating assembly of a RecA homolog, Rad51, into nucleoprotein filaments. The meiosis-specific RecA-homolog Dmc1 does not show this dependence, however. By exploiting the RAD52 independence of Dmc1, we reveal that RAD52 promotes postinvasion steps of both crossover and noncrossover pathways of meiotic recombination in Saccharomyces cerevisiae. This activity resides in the N-terminal region of RAD52, which can anneal complementary DNA strands, and is independent of its Rad51-assembly function. Our findings show that RAD52 functions in temporally and biochemically distinct reactions and suggest a general annealing mechanism for reuniting DSB ends during recombination.
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in vivo assembly and disassembly of rad51 and RAD52 complexes during double strand break repair
The EMBO Journal, 2004Co-Authors: Toshiko Miyazaki, Miki Shinohara, Akira Shinohara, James E. Haber, Debra A BressanAbstract:Assembly and disassembly of Rad51 and RAD52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and RAD52 foci colocalize with a TetR–GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, we observe three distinct foci that colocalize with adjacent GFP chromosome marks. We compared the kinetics of focus formation with recombination intermediates and products when HO-cleaved MATα recombines with the donor, MATa. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATa. We present evidence for three distinct roles for RAD52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and RAD52.
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The N-Terminal DNA-Binding Domain of RAD52 Promotes RAD51-Independent Recombination in Saccharomyces cerevisiae
Genetics, 2003Co-Authors: Mariko Tsukamoto, Miki Shinohara, Kentaro Yamashita, Toshiko Miyazaki, Akira ShinoharaAbstract:In Saccharomyces cerevisiae, the RAD52 protein plays a role in both RAD51-dependent and RAD51-independent recombination pathways. We characterized a RAD52 mutant, RAD52-329, which lacks the C-terminal Rad51-interacting domain, and studied its role in RAD51-independent recombination. The RAD52-329 mutant is completely defective in mating-type switching, but partially proficient in recombination between inverted repeats. We also analyzed the effect of the RAD52-329 mutant on telomere recombination. Yeast cells lacking telomerase maintain telomere length by recombination. The RAD52-329 mutant is deficient in RAD51-dependent telomere recombination, but is proficient in RAD51-independent telomere recombination. In addition, we examined the roles of other recombination genes in the telomere recombination. The RAD51-independent recombination in the RAD52-329 mutant is promoted by a paralogue of RAD52, Rad59. All components of the Rad50-Mre11-Xrs2 complex are also important, but not essential, for RAD51-independent telomere recombination. Interestingly, RAD51 inhibits the RAD51-independent, RAD52-dependent telomere recombination. These findings indicate that RAD52 itself, and more precisely its N-terminal DNA-binding domain, promote an essential reaction in recombination in the absence of RAD51.
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Homologous Recombination, but Not DNA Repair, Is Reduced in Vertebrate Cells Deficient in RAD52
Molecular and cellular biology, 1998Co-Authors: Yuko Yamaguchi-iwai, Minoru Takata, Akira Shinohara, Eiichiro Sonoda, Jean-marie Buerstedde, Olga Bezzubova, Ciaran G. Morrison, Shunichi TakedaAbstract:RAD52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and RAD52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and RAD52 act cooperatively. To define the role of RAD52 in vertebrates, we generated RAD52(-/-) mutants of the chicken B-cell line DT40. Surprisingly, RAD52(-/-) cells were not hypersensitive to DNA damages induced by gamma-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52(-/-) cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52(-/-) cells, showing a clear role for RAD52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.
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RAD52 associates with rpa and functions with rad55 and rad57 to assemble meiotic recombination complexes
Genes & Development, 1998Co-Authors: Stephen L Gasior, Akira Shinohara, Anthony K Wong, Yoshiteru Kora, Douglas K BishopAbstract:We show that the Saccharomyces cerevisiae recombination protein RAD52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of RAD52 and RPA and more limited colocalization of RAD52 with the strand exchange protein Rad51. RAD52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, RAD52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; RAD52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes.
Hitoshi Kurumizaka - One of the best experts on this subject based on the ideXlab platform.
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Functional analyses of the C-terminal half of the Saccharomyces cerevisiae RAD52 protein
Nucleic acids research, 2013Co-Authors: Wataru Kagawa, Takehiko Shibata, Naoto Arai, Kengo Saito, Yuichi Ichikawa, Shusei Sugiyama, Mika Saotome, Hitoshi KurumizakaAbstract:The Saccharomyces cerevisiae RAD52 protein is essential for efficient homologous recombination (HR). An important role of RAD52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, RAD52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although RAD52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of RAD52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of RAD52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The RAD52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of RAD52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of RAD52 and probably play an important role in the mediator activity.
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Vital Roles of the Second DNA-binding Site of RAD52 Protein in Yeast Homologous Recombination
The Journal of biological chemistry, 2011Co-Authors: Naoto Arai, Hitoshi Kurumizaka, Wataru Kagawa, Kengo Saito, Yoshinori Shingu, Tsutomu Mikawa, Takehiko ShibataAbstract:RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a "recombination mediator" to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. RAD52 is the prototype of recombination mediators, and the human RAD52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast RAD52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of RAD52 and Rad51 within the complex are unknown. In the present study, we constructed yeast RAD52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human RAD52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast RAD52 function in vivo. Rad51-RAD52 complexes consisting of these RAD52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that RAD52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing.
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The putative nuclear localization signal of the human RAD52 protein is a potential sumoylation site.
Journal of biochemistry, 2010Co-Authors: Kengo Saito, Wataru Kagawa, Shigeyuki Yokoyama, Takehiro Suzuki, Hidekazu Suzuki, Hisato Saitoh, Satoshi Tashiro, Naoshi Dohmae, Hitoshi KurumizakaAbstract:RAD52, a key factor in homologous recombination (HR), plays important roles in both RAD51-dependent and -independent HR pathways. Several studies have suggested a link between the functional regulation of RAD52 and the protein modification by a small ubiquitin-like modifier (SUMO). However, the molecular mechanism underlying the regulation of RAD52 by SUMO is unknown. To begin investigating this mechanism, we identified possible target sites for sumoylation in the human RAD52 protein by preparing a RAD52-SUMO complex using an established Escherichia coli sumoylation system. Mass spectrometry and amino acid sequencing of the enzymatically digested fragments of the purified complex revealed that the putative nuclear localization signal located near the C terminus of RAD52 was sumoylated. Biochemical studies of the RAD52-SUMO complex suggested that sumoylation at the identified site has no apparent effect on the DNA binding, D-loop formation, ssDNA annealing and RAD51-binding activities of RAD52. On the other hand, visualization of the GFP-fused RAD52 protein in the human cell that contained mutations at the identified sumoylation sites showed clear differences in the cytosolic and nuclear distributions of the protein. These results suggest the possibility of sumoylation playing an important role in the nuclear transport of RAD52.
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Identification of a Second DNA Binding Site in the Human RAD52 Protein
The Journal of biological chemistry, 2008Co-Authors: Wataru Kagawa, Shukuko Ikawa, Hitoshi Kurumizaka, Takehiko Shibata, Kengo Saito, Ako Kagawa, Shigeyuki YokoyamaAbstract:RAD52 plays essential roles in homology-dependent double-strand break repair. Various studies have established the functions of RAD52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of RAD52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within RAD52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the RAD52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that RAD52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that RAD52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.
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homologous pairing activity of the human dna repair proteins xrcc3 rad51c
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Hitoshi Kurumizaka, Maki Nakada, Shukuko Ikawa, Wataru Kagawa, Shunichi Takeda, Shigeyuki Yokoyama, Minoru Takata, Takehiko ShibataAbstract:Abstract The human Xrcc3 protein is involved in the repair of damaged DNA through homologous recombination, in which homologous pairing is a key step. The Rad51 protein is believed to be the only protein factor that promotes homologous pairing in recombinational DNA repair in mitotic cells. In the brain, however, Rad51 expression is extremely low, whereas XRCC3, a human homologue of Saccharomyces cerevisiae RAD57 that activates the Rad51-dependent homologous pairing with the yeast Rad55 protein, is expressed. In this study, a two-hybrid analysis conducted with the use of a human brain cDNA library revealed that the major Xrcc3-interacting protein is a Rad51 paralog, Rad51C/Rad51L2. The purified Xrcc3⋅Rad51C complex, which shows apparent 1:1 stoichiometry, was found to catalyze the homologous pairing. Although the activity is reduced, the Rad51C protein alone also catalyzed homologous pairing, suggesting that Rad51C is a catalytic subunit for homologous pairing. The DNA-binding activity of Xrcc3⋅Rad51C was drastically decreased in the absence of Xrcc3, indicating that Xrcc3 is important for the DNA binding of Xrcc3⋅Rad51C. Electron microscopic observations revealed that Xrcc3⋅Rad51C and Rad51C formed similar filamentous structures with circular single-stranded DNA.
Takehiko Shibata - One of the best experts on this subject based on the ideXlab platform.
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Functional analyses of the C-terminal half of the Saccharomyces cerevisiae RAD52 protein
Nucleic acids research, 2013Co-Authors: Wataru Kagawa, Takehiko Shibata, Naoto Arai, Kengo Saito, Yuichi Ichikawa, Shusei Sugiyama, Mika Saotome, Hitoshi KurumizakaAbstract:The Saccharomyces cerevisiae RAD52 protein is essential for efficient homologous recombination (HR). An important role of RAD52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, RAD52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although RAD52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of RAD52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of RAD52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The RAD52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of RAD52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of RAD52 and probably play an important role in the mediator activity.
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Vital Roles of the Second DNA-binding Site of RAD52 Protein in Yeast Homologous Recombination
The Journal of biological chemistry, 2011Co-Authors: Naoto Arai, Hitoshi Kurumizaka, Wataru Kagawa, Kengo Saito, Yoshinori Shingu, Tsutomu Mikawa, Takehiko ShibataAbstract:RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a "recombination mediator" to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. RAD52 is the prototype of recombination mediators, and the human RAD52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast RAD52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of RAD52 and Rad51 within the complex are unknown. In the present study, we constructed yeast RAD52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human RAD52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast RAD52 function in vivo. Rad51-RAD52 complexes consisting of these RAD52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that RAD52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing.
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Identification of a Second DNA Binding Site in the Human RAD52 Protein
The Journal of biological chemistry, 2008Co-Authors: Wataru Kagawa, Shukuko Ikawa, Hitoshi Kurumizaka, Takehiko Shibata, Kengo Saito, Ako Kagawa, Shigeyuki YokoyamaAbstract:RAD52 plays essential roles in homology-dependent double-strand break repair. Various studies have established the functions of RAD52 in Rad51-dependent and Rad51-independent repair processes. However, the precise molecular mechanisms of RAD52 in these processes remain unknown. In the present study we have identified a novel DNA binding site within RAD52 by a structure-based alanine scan mutagenesis. This site is closely aligned with the putative single-stranded DNA binding site determined previously. Mutations in this site impaired the ability of the RAD52-single-stranded DNA complex to form a ternary complex with double-stranded DNA and subsequently catalyze the formation of D-loops. We found that RAD52 introduces positive supercoils into double-stranded DNA and that the second DNA binding site is essential for this activity. Our findings suggest that RAD52 aligns two recombining DNA molecules within the first and second DNA binding sites to stimulate the homology search and strand invasion processes.
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Heteroduplex joint formation by a stoichiometric complex of Rad51 and RAD52 of Saccharomyces cerevisiae.
The Journal of biological chemistry, 2005Co-Authors: Naoto Arai, Takehiko Shibata, Daisuke Ito, Tadashi Inoue, Hideo TakahashiAbstract:Both Rad51 and RAD52 are required for homologous genetic recombination in Saccharomyces cerevisiae. Rad51 promotes heteroduplex joint formation, a general step in homologous recombination. RAD52 facilitates the binding of Rad51 to replication protein A (RPA)-coated single-stranded DNA. The requirement of RPA can be avoided in vitro, if the single-stranded DNA is short. Using short single-stranded DNA and homologous double-stranded DNA, in the absence of RPA, we found that RAD52 (optimal at three per Rad51) was still required for Rad51-promoted heteroduplex joint formation in vitro, as assayed by the formation of D-loops, suggesting another role for RAD52. Rad51 has to bind to the single-stranded DNA before the addition of double-stranded DNA for efficient D-loop formation. Immunoprecipitation and single-stranded DNA-bead precipitation analyses revealed the presence of the free and DNA-bound complexes of Rad51 and RAD52 at a 1 to 2 stoichiometry. In the presence of single-stranded DNA, in addition to Rad51, RAD52 was required for extensive untwisting that is an intermediate step toward D-loop formation. Thus, these results suggest that the formation of the stoichiometric complex of RAD52 with Rad51 on single-stranded DNA is required for the functional binding of the protein-single-stranded DNA complex to the double-stranded DNA to form D-loops.
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homologous pairing activity of the human dna repair proteins xrcc3 rad51c
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Hitoshi Kurumizaka, Maki Nakada, Shukuko Ikawa, Wataru Kagawa, Shunichi Takeda, Shigeyuki Yokoyama, Minoru Takata, Takehiko ShibataAbstract:Abstract The human Xrcc3 protein is involved in the repair of damaged DNA through homologous recombination, in which homologous pairing is a key step. The Rad51 protein is believed to be the only protein factor that promotes homologous pairing in recombinational DNA repair in mitotic cells. In the brain, however, Rad51 expression is extremely low, whereas XRCC3, a human homologue of Saccharomyces cerevisiae RAD57 that activates the Rad51-dependent homologous pairing with the yeast Rad55 protein, is expressed. In this study, a two-hybrid analysis conducted with the use of a human brain cDNA library revealed that the major Xrcc3-interacting protein is a Rad51 paralog, Rad51C/Rad51L2. The purified Xrcc3⋅Rad51C complex, which shows apparent 1:1 stoichiometry, was found to catalyze the homologous pairing. Although the activity is reduced, the Rad51C protein alone also catalyzed homologous pairing, suggesting that Rad51C is a catalytic subunit for homologous pairing. The DNA-binding activity of Xrcc3⋅Rad51C was drastically decreased in the absence of Xrcc3, indicating that Xrcc3 is important for the DNA binding of Xrcc3⋅Rad51C. Electron microscopic observations revealed that Xrcc3⋅Rad51C and Rad51C formed similar filamentous structures with circular single-stranded DNA.
