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1.
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.  相似文献   

2.
The eukaryotic single-stranded DNA-binding protein, replication protein A (RPA), is essential for DNA replication, and plays important roles in DNA repair and DNA recombination. Rad52 and RPA, along with other members of the Rad52 epistasis group of genes, repair double-stranded DNA breaks (DSBs). Two repair pathways involve RPA and Rad52, homologous recombination and single-strand annealing. Two binding sites for Rad52 have been identified on RPA. They include the previously identified C-terminal domain (CTD) of RPA32 (residues 224-271) and the newly identified domain containing residues 169-326 of RPA70. A region on Rad52, which includes residues 218-303, binds RPA70 as well as RPA32. The N-terminal region of RPA32 does not appear to play a role in the formation of the RPA:Rad52 complex. It appears that the RPA32CTD can substitute for RPA70 in binding Rad52. Sequence homology between RPA32 and RPA70 was used to identify a putative Rad52-binding site on RPA70 that is located near DNA-binding domains A and B. Rad52 binding to RPA increases ssDNA affinity significantly. Mutations in DBD-D on RPA32 show that this domain is primarily responsible for the ssDNA binding enhancement. RPA binding to Rad52 inhibits the higher-order self-association of Rad52 rings. Implications for these results for the "hand-off" mechanism between protein-protein partners, including Rad51, in homologous recombination and single-strand annealing are discussed.  相似文献   

3.
Rad51 protein controls Rad52-mediated DNA annealing   总被引:1,自引:0,他引:1  
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.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
The Xrcc2 and Rad51D/Rad51L3 proteins, which belong to the Rad51 paralogs, are required for homologous recombinational repair (HRR) in vertebrates. The Xrcc2 and Rad51D/Rad51L3 genes, whose products interact with each other, have essential roles in ensuring normal embryonic development. In the present study, we coexpressed the human Xrcc2 and Rad51D/Rad51L3 proteins (Xrcc2 and Rad51D, respectively) in Escherichia coli, and purified the Xrcc2*Rad51D complex to homogeneity. The Xrcc2 small middle dotRad51D complex catalyzed homologous pairing between single-stranded and double-stranded DNA, similar to the function of the Xrcc3*Rad51C complex, which is another complex of the Rad51 paralogs. An electron microscopic analysis showed that Xrcc2*Rad51D formed a multimeric ring structure in the absence of DNA. In the presence of ssDNA, Xrcc2*Rad51D formed a filamentous structure, which is commonly observed among the human homologous pairing proteins, Rad51, Rad52, and Xrcc3*Rad51C.  相似文献   

7.
Human Rad51 protein (HsRad51) is a homolog of Escherichia coli RecA protein, and functions in DNA repair and recombination. In higher eukaryotes, Rad51 protein is essential for cell viability. The N-terminal region of HsRad51 is highly conserved among eukaryotic Rad51 proteins but is absent from RecA, suggesting a Rad51-specific function for this region. Here, we have determined the structure of the N-terminal part of HsRad51 by NMR spectroscopy. The N-terminal region forms a compact domain consisting of five short helices, which shares structural similarity with a domain of endonuclease III, a DNA repair enzyme of E. coli. NMR experiments did not support the involvement of the N-terminal domain in HsRad51-HsBrca2 interaction or the self-association of HsRad51 as proposed by previous studies. However, NMR tiration experiments demonstrated a physical interaction of the domain with DNA, and allowed mapping of the DNA binding surface. Mutation analysis showed that the DNA binding surface is essential for double-stranded and single-stranded DNA binding of HsRad51. Our results suggest the presence of a DNA binding site on the outside surface of the HsRad51 filament and provide a possible explanation for the regulation of DNA binding by phosphorylation within the N-terminal domain.  相似文献   

8.
Error-free repair by homologous recombination of DNA double-strand breaks induced by ionizing radiation (IR) requires the Rad52 group proteins, including Rad51 and Rad54, in the yeast Saccharomyces cerevisiae [1]. The formation of a 'joint' molecule between the damaged DNA and the homologous repair template is a key step in recombination mediated by Rad51 and stimulated by Rad54 [2] [3] [4] [5]. Mammalian homologs of Rad51 and Rad54 have been identified [2] [3] [6]. Here, we demonstrate that mouse Rad54 (mRad54) formed IR-induced nuclear foci that colocalized with mRad51. Interaction between mRad51 and mRad54 was induced by genotoxic stress, but only when lesions that required mRad54 for their repair were formed. Interestingly, mRad54 was essential for the formation of IR-induced mRad51 foci. Rad54 belongs to the SWI2/SNF2 protein family, members of which modulate protein-DNA interactions in an ATP-driven manner [7]. Results of a topological assay suggested that purified human Rad54 (hRad54) protein can unwind double-stranded (ds) DNA at the expense of ATP hydrolysis. Unwinding of the homologous repair template could promote the formation or stabilization of hRad51-mediated joint molecules. Rad54 appears to be required downstream of other Rad52 group proteins, such as Rad52 and the Rad55-Rad57 heterodimer, that assist Rad51 in interacting with the broken DNA [2] [3] [4].  相似文献   

9.
Yeast Rad51 promotes homologous pairing and strand exchange in vitro, but this activity is inefficient in the absence of the accessory proteins, RPA, Rad52, Rad54 and the Rad55-Rad57 heterodimer. A class of rad51 alleles was isolated that suppresses the requirement for RAD55 and RAD57 in DNA repair, but not the other accessory factors. Five of the six mutations isolated map to the region of Rad51 that by modeling with RecA corresponds to one of the DNA-binding sites. The other mutation is in the N-terminus of Rad51 in a domain implicated in protein-protein interactions and DNA binding. The Rad51-I345T mutant protein shows increased binding to single- and double-stranded DNA, and is proficient in displacement of replication protein A (RPA) from single-stranded DNA, suggesting that the normal function of Rad55-Rad57 is promotion and stabilization of Rad51-ssDNA complexes.  相似文献   

10.
An essential role of DmRad51/SpnA in DNA repair and meiotic checkpoint control   总被引:13,自引:0,他引:13  
Rad51 is a conserved protein essential for recombinational repair of double-stranded DNA breaks (DSBs) in somatic cells and during meiosis in germ cells. Yeast Rad51 mutants are viable but show meiosis defects. In the mouse, RAD51 deletions cause early embryonic death, suggesting that in higher eukaryotes Rad51 is required for viability. Here we report the identification of SpnA as the Drosophila Rad51 gene, whose sequence among the five known Drosophila Rad51-like genes is most closely related to the Rad51 homologs of human and yeast. DmRad51/spnA null mutants are viable but oogenesis is disrupted by the activation of a meiotic recombination checkpoint. We show that the meiotic phenotypes result from an inability to effectively repair DSBs. Our study further demonstrates that in Drosophila the Rad51-dependent homologous recombination pathway is not essential for DNA repair in the soma, unless exposed to DNA damaging agents. We therefore propose that under normal conditions a second, Rad51-independent, repair pathway prevents the lethal effects of DNA damage.  相似文献   

11.
The RAD52 gene is essential for homologous recombination in the yeast Saccharomyces cerevisiae. RAD52 is the archetype in an epistasis group of genes essential for DNA damage repair. By catalyzing the replacement of replication protein A with Rad51 on single-stranded DNA, Rad52 likely promotes strand invasion of a double-stranded DNA molecule by single-stranded DNA. Although the sequence and in vitro functions of mammalian RAD52 are conserved with those of yeast, one difference is the presence of introns and consequent splicing of the mammalian RAD52 pre-mRNA. We identified two novel splice variants from the RAD52 gene that are expressed in adult mouse tissues. Expression of these splice variants in tissue culture cells elevates the frequency of recombination that uses a sister chromatid template. To characterize this dominant phenotype further, the RAD52 gene from the yeast Saccharomyces cerevisiae was truncated to model the mammalian splice variants. The same dominant sister chromatid recombination phenotype seen in mammalian cells was also observed in yeast. Furthermore, repair from a homologous chromatid is reduced in yeast, implying that the choice of alternative repair pathways may be controlled by these variants. In addition, a dominant DNA repair defect induced by one of the variants in yeast is suppressed by overexpression of RAD51, suggesting that the Rad51-Rad52 interaction is impaired.  相似文献   

12.
Homologous recombination is important for the repair of double-stranded DNA breaks in all organisms. Rad51 and Rad54 proteins are two key components of the homologous recombination machinery in eukaryotes. In vitro, Rad51 protein assembles with single-stranded DNA to form the helical nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. Here, using physical criteria, we demonstrate directly the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. Thus, the co-assembly of an interacting partner with the Rad51 nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein.  相似文献   

13.
In the yeast Saccharomyces cerevisiae, the RAD52 gene is indispensable for homologous recombination and DNA repair. Rad52 protein binds DNA, anneals complementary ssDNA strands, and self-associates to form multimeric complexes. Moreover, Rad52 physically interacts with the Rad51 recombinase and serves as a mediator in the Rad51-catalyzed DNA strand exchange reaction. Here, we examine the functional significance of the Rad51/Rad52 interaction. Through a series of deletions, we have identified residues 409-420 of Rad52 as being indispensable and likely sufficient for its interaction with Rad51. We have constructed a four-amino acid deletion mutation within this region of Rad52 to ablate its interaction with Rad51. We show that the rad52delta409-412 mutant protein is defective in the mediator function in vitro even though none of the other Rad52 activities, namely, DNA binding, ssDNA annealing, and protein oligomerization, are affected. We also show that the sensitivity of the rad52delta409-412 mutant to ionizing radiation can be complemented by overexpression of Rad51. These results thus demonstrate the significance of the Rad51-Rad52 interaction in homologous recombination.  相似文献   

14.
Rad51, Rad52, and replication protein-A (RPA) play crucial roles in the repair of DNA double-strand breaks in Saccharomyces cerevisiae. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 recruits Rad51 into single-stranded DNAs (ssDNAs) that are saturated with RPA. Rad52 also promotes annealing of ssDNA strands that are complexed with RPA. Specific protein-protein interactions are involved in these reactions. Here we report new biochemical characteristics of these protein interactions. First, Rad52-RPA interaction requires multiple molecules of RPA to be associated with ssDNA, suggesting that multiple contacts between the Rad52 ring and RPA-ssDNA filament are needed for stable binding. Second, RPA-t11, which is a recombination-deficient mutant of RPA, displays a defect in interacting with Rad52 in the presence of salt above 50 mM, explaining the defect in Rad52-mediated ssDNA annealing in the presence of this mutation. Third, ssDNA annealing promoted by Rad52 is preceded by aggregation of multiple RPA-ssDNA complexes with Rad52, and Rad51 inhibits this aggregation. These results suggest a regulatory role for Rad51 that suppresses ssDNA annealing and facilitates DNA strand invasion. Finally, the Rad51-double-stranded DNA complex disrupts Rad52-RPA interaction in ssDNA and titrates Rad52 from RPA. This suggests an additional regulatory role for Rad51 following DNA strand invasion, where Rad51-double-stranded DNA may inhibit illegitimate second-end capture to ensure the error-free repair of a DNA double-strand break.  相似文献   

15.
Recombination is important for the repair of DNA damage and for chromosome segregation during meiosis; it has also been shown to participate in the regulation of cell proliferation. In the yeast Saccharomyces cerevisiae, recombination requires products of the RAD52 epistasis group. The Rad51 protein associates with the Rad51, Rad52, Rad54, and Rad55 proteins to form a dynamic complex. We describe a new strategy to screen for mutations which cause specific disruption of the interaction between certain proteins in the complex, leaving other interactions intact. This approach defines distinct protein interaction domains and protein relationships within the Rad51 complex. Alignment of the mutations onto the constructed three-dimensional model of the Rad51 protein reveal possible partially overlapping interfaces for the Rad51-Rad52 and the Rad51-Rad54 interactions. Rad51-Rad55 and Rad51-Rad51 interactions are affected by the same spectrum of mutations, indicating similarity between the two modes of binding. Finally, the detection of a subset of mutations within Rad51 which disrupt the interaction with mutant Rad52 protein but activate the interaction with Rad54 suggests that dynamic changes within the Rad51 protein may contribute to an ordered reaction process.  相似文献   

16.
The genes of the Saccharomyces cerevisiae RAD52 epistasis group are required for the repair of ionizing radiation-induced DNA damage. Three of these genes, RAD51, RAD55, and RAD57, have been identified as putative RecA homologs. An important feature of RecA is its ability to bind and hydrolyze ATP. RAD55 and RAD57 contain putative nucleotide binding motifs, and the importance of these motifs was determined by constructing site-directed mutations of the conserved lysine residue within the Walker A-box. Changing the lysine residue to arginine or alanine resulted in a mutant phenotype in DNA repair and sporulation for Rad55 but not for Rad57. Protein-protein interactions among Rad51, Rad55, and Rad57 were tested for by the two-hybrid system. Rad55 was shown to interact with Rad51 and Rad57 but not with itself. Additionally, no interaction between Rad57 and Rad51 or between Rad57 and itself was detected. Consistent with the hypothesis that Rad55 and Rad57 may function within, or stabilize, a protein complex, we found that RAD51 expressed from a high-copy-number plasmid suppresses the DNA repair defect of strains carrying rad55 and rad57 mutations. These data, in conjunction with other reports, demonstrate the importance of protein-protein interactions in the process of DNA repair.  相似文献   

17.
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.  相似文献   

18.
The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.  相似文献   

19.
Meiotic homologous recombination in Saccharomyces cerevisiae involves formation of nucleoprotein filaments of Rad51 and Dmc1 that mediate DNA strand exchange between homologous chromosomes. The Mei5-Sae3 protein complex functions as a recombination mediator to promote nucleation of the Dmc1 recombinase onto replication protein A-coated single-stranded DNA. Here, we have expressed and purified the Mei5 protein, Sae3 protein and the Mei5-Sae3 complex for biochemical studies. We show the Mei5-Sae3 complex preferentially binds a fork-like DNA substrate to 3' overhanging DNA, single-stranded DNA or double-stranded DNA. We demonstrate that Mei5 confers DNA binding activity to the Mei5-Sae3 complex. We determined Mei5-Sae3 interacts with the Rad51 recombinase through the N-terminal domain of Mei5. Unlike Rad52, Mei5-Sae3 lacks recombination mediator activity for Rad51. Importantly, we find that the Mei5-Sae3 complex does not harbor single-strand DNA annealing activity. These properties of the Mei5-Sae3 complex distinguishes it from the Rad52 protein, which serves as the mediator of Rad51 and is involved in the single-strand DNA annealing pathway of homologous recombination.  相似文献   

20.
Yeast rad50 and mre11 nuclease mutants are hypersensitive to physical and chemical agents that induce DNA double-strand breaks (DSBs). This sensitivity was suppressed by elevating intracellular levels of TLC1, the RNA subunit of telomerase. Suppression required proteins linked to homologous recombination, including Rad51, Rad52, Rad59 and Exo1, but not genes of the nonhomologous end-joining (NHEJ) repair pathway. Deletion mutagenesis experiments demonstrated that the 5'-end of TLC1 RNA was essential and a segment containing a binding site for the Yku70/Yku80 complex was sufficient for suppression. A mutant TLC1 RNA unable to associate with Yku80 protein did not increase resistance. These and other genetic studies indicated that association of the Ku heterodimer with broken DNA ends inhibits recombination in mrx mutants, but not in repair-proficient cells or in other DNA repair single mutants. In support of this model, DNA damage resistance of mrx cells was enhanced when YKU70 was co-inactivated. Defective recombinational repair of DSBs in mrx cells thus arises from at least two separate processes: loss of Mrx nuclease-associated DNA end-processing and inhibition of the Exo1-mediated secondary recombination pathway by Ku.  相似文献   

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