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Wolf Dietrich Heyer - One of the best experts on this subject based on the ideXlab platform.
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terminal association of rad54 protein with the RAD51 dsdna filament
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Konstantin Kiianitsa, Jachen A Solinger, Wolf Dietrich HeyerAbstract:Rad54 protein is a Snf2-related dsDNA-specific ATPase essential for homologous recombination mediated by RAD51 protein, the eukaryotic RecA ortholog. Snf2-related enzymes couple ATP hydrolysis with translocation on dsDNA to remodel or dissociate a wide variety of protein–dsDNA complexes. Rad54 and RAD51 interact through species-specific contacts and mutually stimulate their biochemical activities. Specifically, RAD51 bound to dsDNA, the product of homologous recombination after DNA-strand exchange, stimulates the Rad54 ATPase up to 6-fold, leading to the turnover of RAD51 in the product complex. Electron microscopy visualized the RAD51–Rad54 interaction on dsDNA, showing that an oligomeric form of Rad54 associates preferentially with termini of the RAD51–dsDNA filament. Our data support a mechanism of processive dsDNA–RAD51 filament dissociation by the translocating Rad54 protein.
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rad54 a swi2 snf2 like recombinational repair protein disassembles RAD51 dsdna filaments
Molecular Cell, 2002Co-Authors: Jachen A Solinger, Konstantin Kiianitsa, Wolf Dietrich HeyerAbstract: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.
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spontaneous and double strand break induced recombination and gene conversion tract lengths are differentially affected by overexpression of wild type or atpase defective yeast rad54
Nucleic Acids Research, 2002Co-Authors: Perry M Kim, Jachen A Solinger, Wolf Dietrich Heyer, Kimberly S Paffett, Jac A NickoloffAbstract:Rad54 plays key roles in homologous recombination (HR) and double-strand break (DSB) repair in yeast, along with RAD51, Rad52, Rad55 and Rad57. Rad54 belongs to the Swi2/Snf2 family of DNA-stimulated ATPases. RAD51 nucleoprotein filaments catalyze DNA strand exchange and Rad54 augments this activity of RAD51. Mutations in the Rad54 ATPase domain (ATPase–) impair Rad54 function in vitro, sensitize yeast to killing by methylmethane sulfonate and reduce spontaneous gene conversion. We found that overexpression of ATPase– Rad54 reduced spontaneous direct repeat gene conversion and increased both spontaneous direct repeat deletion and spontaneous allelic conversion. Overexpression of ATPase– Rad54 decreased DSB-induced allelic conversion, but increased chromosome loss and DSB-dependent lethality. Thus, ATP hydrolysis by Rad54 contributes to genome stability by promoting high-fidelity DSB repair and suppressing spontaneous deletions. Overexpression of wild-type Rad54 did not alter DSB-induced HR levels, but conversion tract lengths were reduced. Interestingly, ATPase– Rad54 decreased overall HR levels and increased tract lengths. These tract length changes provide new in vivo evidence that Rad54 functions in the post-synaptic phase during recombinational repair of DSBs.
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rad54 protein stimulates heteroduplex dna formation in the synaptic phase of dna strand exchange via specific interactions with the presynaptic RAD51 nucleoprotein filament
Journal of Molecular Biology, 2001Co-Authors: Jachen A Solinger, Tomohiko Sugiyama, Wolf Dietrich Heyer, Geraldine Lutz, Stephen C KowalczykowskiAbstract: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.
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rad54 protein is targeted to pairing loci by the RAD51 nucleoprotein filament
Molecular Cell, 2000Co-Authors: Alexander V. Mazin, Carole J Bornarth, Jachen A Solinger, Wolf Dietrich HeyerAbstract:RAD51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. RAD51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature RAD51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to RAD51 within the filament. Reciprocally, the RAD51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the RAD51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.
Alexander V. Mazin - One of the best experts on this subject based on the ideXlab platform.
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rad52 inverse strand exchange drives rna templated dna double strand break repair
Molecular Cell, 2017Co-Authors: Olga M Mazina, Kritika Hanamshet, Havva Keskin, Francesca Storici, Alexander V. MazinAbstract:RNA can serve as a template for DNA double-strand break repair in yeast cells, and Rad52, a member of the homologous recombination pathway, emerged as an important player in this process. However, the exact mechanism of how Rad52 contributes to RNA-dependent DSB repair remained unknown. Here, we report an unanticipated activity of yeast and human Rad52: inverse strand exchange, in which Rad52 forms a complex with dsDNA and promotes strand exchange with homologous ssRNA or ssDNA. We show that in eukaryotes, inverse strand exchange between homologous dsDNA and RNA is a distinctive activity of Rad52; neither RAD51 recombinase nor the yeast Rad52 paralog Rad59 has this activity. In accord with our in vitro results, our experiments in budding yeast provide evidence that Rad52 inverse strand exchange plays an important role in RNA-templated DSB repair in vivo.
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RAD51 Protein Stimulates the Branch Migration Activity of Rad54 Protein
The Journal of biological chemistry, 2008Co-Authors: Matthew J. Rossi, Alexander V. MazinAbstract:The RAD51 and Rad54 proteins play important roles during homologous recombination in eukaryotes. RAD51 forms a nucleoprotein filament on single-stranded DNA and performs the initial steps of double strand break repair. Rad54 belongs to the Swi2/Snf2 family of ATP-dependent DNA translocases. We previously showed that Rad54 promotes branch migration of Holliday junctions. Here we find that human RAD51 (hRAD51) significantly stimulates the branch migration activity of hRad54. The stimulation appears to be evolutionarily conserved, as yeast RAD51 also stimulates the branch migration activity of yeast Rad54. We further investigated the mechanism of this stimulation. Our results demonstrate that the stimulation of hRad54-promoted branch migration by hRAD51 is driven by specific protein-protein interactions, and the active form of the hRAD51 filament is more stimulatory than the inactive one. The current results support the hypothesis that the hRAD51 conformation state has a strong effect on interaction with hRad54 and ultimately on the function of hRad54 in homologous recombination.
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ca2 activates human homologous recombination protein RAD51 by modulating its atpase activity
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Dmitry V Bugreev, Alexander V. MazinAbstract:Human RAD51 (hRAD51) protein plays a key role in homologous recombination and DNA repair. hRAD51 protein forms a helical filament on single-stranded DNA (ssDNA), which performs the basic steps of homologous recombination: a search for homologous double-stranded DNA (dsDNA) and DNA strand exchange. hRAD51 protein possesses DNA-dependent ATPase activity; however, the role of this activity has not been understood. Our current results show that Ca2+ greatly stimulates DNA strand exchange activity of hRAD51 protein. We found that Ca2+ exerts its stimulatory effect by modulating the ATPase activity of hRAD51 protein. Our data demonstrate that, in the presence of Mg2+, the hRAD51-ATP-ssDNA filament is quickly converted to an inactive hRAD51-ADP-ssDNA form, due to relatively rapid ATP hydrolysis and slow dissociation of ADP. Ca2+ maintains the active hRAD51-ATP-ssDNA filament by reducing the ATP hydrolysis rate. These findings demonstrate a crucial role of the ATPase activity in regulation of DNA strand exchange activity of hRAD51 protein. This mechanism of RAD51 protein regulation by modulating its ATPase activity is evolutionarily recent; we found no such mechanism for yeast RAD51 (yRAD51) protein.
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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. MazinAbstract: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.
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rad54 protein is targeted to pairing loci by the RAD51 nucleoprotein filament
Molecular Cell, 2000Co-Authors: Alexander V. Mazin, Carole J Bornarth, Jachen A Solinger, Wolf Dietrich HeyerAbstract:RAD51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. RAD51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature RAD51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to RAD51 within the filament. Reciprocally, the RAD51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the RAD51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.
Jachen A Solinger - One of the best experts on this subject based on the ideXlab platform.
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terminal association of rad54 protein with the RAD51 dsdna filament
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Konstantin Kiianitsa, Jachen A Solinger, Wolf Dietrich HeyerAbstract:Rad54 protein is a Snf2-related dsDNA-specific ATPase essential for homologous recombination mediated by RAD51 protein, the eukaryotic RecA ortholog. Snf2-related enzymes couple ATP hydrolysis with translocation on dsDNA to remodel or dissociate a wide variety of protein–dsDNA complexes. Rad54 and RAD51 interact through species-specific contacts and mutually stimulate their biochemical activities. Specifically, RAD51 bound to dsDNA, the product of homologous recombination after DNA-strand exchange, stimulates the Rad54 ATPase up to 6-fold, leading to the turnover of RAD51 in the product complex. Electron microscopy visualized the RAD51–Rad54 interaction on dsDNA, showing that an oligomeric form of Rad54 associates preferentially with termini of the RAD51–dsDNA filament. Our data support a mechanism of processive dsDNA–RAD51 filament dissociation by the translocating Rad54 protein.
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rad54 a swi2 snf2 like recombinational repair protein disassembles RAD51 dsdna filaments
Molecular Cell, 2002Co-Authors: Jachen A Solinger, Konstantin Kiianitsa, Wolf Dietrich HeyerAbstract: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.
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spontaneous and double strand break induced recombination and gene conversion tract lengths are differentially affected by overexpression of wild type or atpase defective yeast rad54
Nucleic Acids Research, 2002Co-Authors: Perry M Kim, Jachen A Solinger, Wolf Dietrich Heyer, Kimberly S Paffett, Jac A NickoloffAbstract:Rad54 plays key roles in homologous recombination (HR) and double-strand break (DSB) repair in yeast, along with RAD51, Rad52, Rad55 and Rad57. Rad54 belongs to the Swi2/Snf2 family of DNA-stimulated ATPases. RAD51 nucleoprotein filaments catalyze DNA strand exchange and Rad54 augments this activity of RAD51. Mutations in the Rad54 ATPase domain (ATPase–) impair Rad54 function in vitro, sensitize yeast to killing by methylmethane sulfonate and reduce spontaneous gene conversion. We found that overexpression of ATPase– Rad54 reduced spontaneous direct repeat gene conversion and increased both spontaneous direct repeat deletion and spontaneous allelic conversion. Overexpression of ATPase– Rad54 decreased DSB-induced allelic conversion, but increased chromosome loss and DSB-dependent lethality. Thus, ATP hydrolysis by Rad54 contributes to genome stability by promoting high-fidelity DSB repair and suppressing spontaneous deletions. Overexpression of wild-type Rad54 did not alter DSB-induced HR levels, but conversion tract lengths were reduced. Interestingly, ATPase– Rad54 decreased overall HR levels and increased tract lengths. These tract length changes provide new in vivo evidence that Rad54 functions in the post-synaptic phase during recombinational repair of DSBs.
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rad54 protein stimulates heteroduplex dna formation in the synaptic phase of dna strand exchange via specific interactions with the presynaptic RAD51 nucleoprotein filament
Journal of Molecular Biology, 2001Co-Authors: Jachen A Solinger, Tomohiko Sugiyama, Wolf Dietrich Heyer, Geraldine Lutz, Stephen C KowalczykowskiAbstract: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.
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rad54 protein is targeted to pairing loci by the RAD51 nucleoprotein filament
Molecular Cell, 2000Co-Authors: Alexander V. Mazin, Carole J Bornarth, Jachen A Solinger, Wolf Dietrich HeyerAbstract:RAD51 and Rad54 proteins are important for the repair of double-stranded DNA (dsDNA) breaks by homologous recombination in eukaryotes. RAD51 assembles on single-stranded DNA (ssDNA) to form a helical nucleoprotein filament that performs homologous pairing with dsDNA; Rad54 stimulates this pairing substantially. Here, we demonstrate that Rad54 acts in concert with the mature RAD51-ssDNA filament. Enhancement of DNA pairing by Rad54 is greatest at an equimolar ratio relative to RAD51 within the filament. Reciprocally, the RAD51-ssDNA filament enhances both the dsDNA-dependent ATPase and the dsDNA unwinding activities of Rad54. We conclude that Rad54 participates in the DNA homology search as a component of the RAD51-nucleoprotein filament and that the filament delivers Rad54 to the dsDNA pairing locus, thereby linking the unwinding of potential target DNA with the homology search process.
Stephen C West - One of the best experts on this subject based on the ideXlab platform.
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role of RAD51c and xrcc3 in genetic recombination and dna repair
Journal of Biological Chemistry, 2007Co-Authors: Madalena Tarsounas, Paul Oregan, Stephen C WestAbstract:Abstract In germ line cells, recombination is required for gene reassortment and proper chromosome segregation at meiosis, whereas in somatic cells it provides an important mechanism for the repair of DNA double-strand breaks. Five proteins (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) that share homology with RAD51 recombinase and are known as the RAD51 paralogs are important for recombinational repair, as paralog-defective cell lines exhibit spontaneous chromosomal aberrations, defective DNA repair, and reduced gene targeting. The paralogs form two distinct protein complexes, RAD51B-RAD51C-RAD51D-XRCC2 and RAD51C-XRCC3, but their precise cellular roles remain unknown. Here, we show that, like MLH1, RAD51C localized to mouse meiotic chromosomes at pachytene/diplotene. Using immunoprecipitation and gel filtration analyses, we found that Holliday junction resolvase activity associated tightly and co-eluted with the 80-kDa RAD51C-XRCC3 complex. Taken together, these data indicate that the RAD51C-XRCC3-associated Holliday junction resolvase complex associates with crossovers and may play an essential role in the resolution of recombination intermediates prior to chromosome segregation.
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telomere maintenance requires the RAD51d recombination repair protein
Cell, 2004Co-Authors: Madalena Tarsounas, Phillip G Smiraldo, Douglas L Pittman, Purificacion Munoz, Andreas Claas, Maria A Blasco, Stephen C WestAbstract:Abstract The five RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are required in mammalian cells for normal levels of genetic recombination and resistance to DNA-damaging agents. We report here that RAD51D is also involved in telomere maintenance. Using immunofluorescence labeling, electron microscopy, and chromatin immunoprecipitation assays, RAD51D was shown to localize to the telomeres of both meiotic and somatic cells. Telomerase-positive RAD51d −/− Trp53 −/− primary mouse embryonic fibroblasts (MEFs) exhibited telomeric DNA repeat shortening compared to Trp53 −/− or wild-type MEFs. Moreover, elevated levels of chromosomal aberrations were detected, including telomeric end-to-end fusions, a signature of telomere dysfunction. Inhibition of RAD51D synthesis in telomerase-negative immortalized human cells by siRNA also resulted in telomere erosion and chromosome fusion. We conclude that RAD51D plays a dual cellular role in both the repair of DNA double-strand breaks and telomere protection against attrition and fusion.
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identification and purification of two distinct complexes containing the five RAD51 paralogs
Genes & Development, 2001Co-Authors: Jeanyves Masson, Fiona E Benson, Madalena Tarsounas, Alicja Z Stasiak, Andrzej Stasiak, Rajvee Shah, Michael J Mcilwraith, Stephen C WestAbstract:Cells defective in any of the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3) are sensitive to DNA cross-linking agents and to ionizing radiation. Because the paralogs are required for the assembly of DNA damage-induced RAD51 foci, and mutant cell lines are defective in homologous recombination and show genomic instability, their defect is thought to be caused by an inability to promote efficient recombinational repair. Here, we show that the five paralogs exist in two distinct complexes in human cells: one contains RAD51B, RAD51C, RAD51D, and XRCC2 (defined as BCDX2), whereas the other consists of RAD51C with XRCC3. Both protein complexes have been purified to homogeneity and their biochemical properties investigated. BCDX2 binds single-stranded DNA and single-stranded gaps in duplex DNA, in accord with the proposal that the paralogs play an early (pre-RAD51) role in recombinational repair. Moreover, BCDX2 complex binds specifically to nicks in duplex DNA. We suggest that the extreme sensitivity of paralog-defective cell lines to cross-linking agents is owing to defects in the processing of incised cross links and the consequential failure to initiate recombinational repair at these sites.
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complex formation by the human RAD51c and xrcc3 recombination repair proteins
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Jeanyves Masson, Fiona E Benson, Alicja Z Stasiak, Stephen C WestAbstract:In vertebrates, the RAD51 protein is required for genetic recombination, DNA repair, and cellular proliferation. Five paralogs of RAD51, known as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, have been identified and also shown to be required for recombination and genome stability. At the present time, however, very little is known about their biochemical properties or precise biological functions. As a first step toward understanding the roles of the RAD51 paralogs in recombination, the human RAD51C and XRCC3 proteins were overexpressed and purified from baculovirus-infected insect cells. The two proteins copurify as a complex, a property that reflects their endogenous association observed in HeLa cells. Purified RAD51C–XRCC3 complex binds single-stranded, but not duplex DNA, to form protein–DNA networks that have been visualized by electron microscopy.
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visualisation of human rad52 protein and its complexes with hRAD51 and dna
Journal of Molecular Biology, 1998Co-Authors: E Van Dyck, Nasser Hajibagheri, Andrzej Stasiak, Stephen C WestAbstract:The human Rad52 protein stimulates joint molecule formation by hRAD51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRAD51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRAD51 invasion and nucleoprotein filament formation. When Rad52-RAD51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the RAD51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human RAD51 and Rad52 recombination/repair proteins.
Joanna S Albala - One of the best experts on this subject based on the ideXlab platform.
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the atpase motif in RAD51d is required for resistance to dna interstrand crosslinking agents and interaction with RAD51c
Mutagenesis, 2005Co-Authors: Aaron M Gruver, Kristi A Miller, Changanamkandath Rajesh, Phillip G Smiraldo, Rachel Balder, Katie M Stiles, Joanna S Albala, Saravanan Kaliyaperumal, Douglas L PittmanAbstract:Homologous recombination (HR) is a mechanism for repairing DNA interstrand crosslinks and double-strand breaks. In mammals, HR requires the activities of the RAD51 family (RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1), each of which contains conserved ATP binding sequences (Walker Motifs A and B). RAD51D is a DNA-stimulated ATPase that interacts directly with RAD51C and XRCC2. To test the hypothesis that ATP binding and hydrolysis by RAD51D are required for the repair of interstrand crosslinks, site-directed mutations in Walker Motif A were generated, and complementation studies were performed in RAD51d-deficient mouse embryonic fibroblasts. The K113R and K113A mutants demonstrated a respective 96 and 83% decrease in repair capacity relative to wild-type. Further examination of these mutants, by yeast two-hybrid analyses, revealed an 8-fold reduction in the ability to associate with RAD51C whereas interaction with XRCC2 was retained at a level similar to the S111T control. These cell-based studies are the first evidence that ATP binding and hydrolysis by RAD51D are required for efficient HR repair of DNA interstrand crosslinks.
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nuclear localization of RAD51b is independent of RAD51c and brca2
Mutagenesis, 2005Co-Authors: Kristi A Miller, L. H. Thompson, Alice N Yamada, John M. Hinz, Joanna S AlbalaAbstract:Human RAD51 is critical for the maintenance of genome stability through its role in the repair of DNA double-strand breaks. RAD51B (RAD51L1/hRec2) is one of the five known paralogs of human RAD51 found in a multi-protein complex with three other RAD51 paralogs, RAD51C, RAD51D and Xrcc2. Examination of EGFP-RAD51B fusion protein in HeLa S3 cells and immunofluorescence in several human cell lines confirms the nuclear localization of RAD51B. This is the first report to detail putative interactions of a RAD51 paralog protein with BRCA2. Utilization of a BRCA2 mutant cell line, CAPAN-1 suggests that RAD51B localizes to the nucleus independent of BRCA2. Although both RAD51B and BRCA2 are clearly involved in the homologous recombinational repair pathway, RAD51B and BRCA2 do not appear to associate directly. Furthermore, mutations in the KKLK motif of RAD51B, amino acid residues 4-7, mislocalizes RAD51B to the cytoplasm suggesting that this is the nuclear localization signal for the RAD51B protein. Examination of wild-type EGFP-RAD51B fusion protein in mammalian cells deficient in RAD51C showed that RAD51B localizes to the nucleus independent of RAD51C; further suggesting that RAD51B, like RAD51C, contains its own nuclear localization signal.
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domain mapping of the RAD51 paralog protein complexes
Nucleic Acids Research, 2004Co-Authors: Kristi A Miller, Dorota Sawicka, Daniel Barsky, Joanna S AlbalaAbstract:The five human RAD51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, RAD51B–RAD51C–RAD51D–Xrcc2 (BCDX2) and RAD51C–Xrcc3 (CX3). Based on the recent Pyrococcus furiosus RAD51 structure, we have used homology modeling to design deletion mutants of the RAD51 paralogs. The models of the human RAD51B, RAD51C, Xrcc3 and murine RAD51D (mRAD51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of RAD51B containing amino acid residues 1–75 interacts with the C-terminus and linker of RAD51C, residues 79–376, and this region of RAD51C also interacts with mRAD51D and Xrcc3. We have also determined that the N-terminal domain of mRAD51D, residues 4–77, binds to Xrcc2 while the C-terminal domain of mRAD51D, residues 77–328, binds RAD51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes.
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RAD51c interacts with RAD51b and is central to a larger protein complex in vivo exclusive of RAD51
Journal of Biological Chemistry, 2002Co-Authors: Kristi A Miller, Daniel M Yoshikawa, Ian R Mcconnell, Robin Clark, David Schild, Joanna S AlbalaAbstract:RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins. Moreover, co-immunoprecipitation of the RAD51B and RAD51C proteins from HeLa, MCF10A, and MCF7 cells strongly suggests the existence of an endogenous RAD51B/RAD51C heterocomplex. We extended these observations to examine the interaction between the RAD51B/RAD51C complex and the other RAD51 paralogs. Immunoprecipitation using protein-specific antibodies showed that RAD51C is central to a single large protein complex and/or several smaller complexes with RAD51B, RAD51D, XRCC2, and XRCC3. However, our experiments showed no evidence for the inclusion of RAD51 within these complexes. Further analysis is required to elucidate the function of the RAD51B/RAD51C heterocomplex and its association with the other RAD51 paralogs in the processes of homologous recombination and DNA double-strand break repair.
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interactions involving the RAD51 paralogs RAD51c and xrcc3 in human cells
Nucleic Acids Research, 2002Co-Authors: Claudia Wiese, L. H. Thompson, David W Collins, Amy Kronenberg, Joanna S Albala, David SchildAbstract:Homologous recombinational repair of DNA double-strand breaks and crosslinks in human cells is likely to require RAD51 and the five RAD51 paralogs (XRCC2, XRCC3, RAD51B/RAD51L1, RAD51C/RAD51L2 and RAD51D/RAD51L3), as has been shown in chicken and rodent cells. Previously, we reported on the interactions among these proteins using baculovirus and two- and three-hybrid yeast systems. To test for interactions involving XRCC3 and RAD51C, stable human cell lines have been isolated that express (His)6-tagged versions of XRCC3 or RAD51C. Ni2+-binding experiments demonstrate that XRCC3 and RAD51C interact in human cells. In addition, we find that RAD51C, but not XRCC3, interacts directly or indirectly with RAD51B, RAD51D and XRCC2. These results argue that there are at least two complexes of RAD51 paralogs in human cells (RAD51C–XRCC3 and RAD51B–RAD51C–RAD51D–XRCC2), both containing RAD51C. Moreover, RAD51 is not found in these complexes. X-ray treatment did not alter either the level of any RAD51 paralog or the observed interactions between paralogs. However, the endogenous level of RAD51C is moderately elevated in the XRCC3-overexpressing cell line, suggesting that dimerization between these proteins might help stabilize RAD51C.
