A first year graduate student, Ann Dee Margulies, changed my research career in 1962 by challenging me to direct her in the isolation of recombination-deficient mutants of Escherichia coli K-12. She succeeded in isolating two mutants, which conjugated with donor strains and received the donor DNA, but could not recombine that DNA with their own chromosomes. Ann Dee showed that both mutants were much more sensitive to UV radiation than was the wild type. Furthermore, she showed that one of these mutants carried a single mutation affecting both recombination and resistance. This work, published in 1965, was the first demonstration of the recA gene of E. coli. Subsequent work led to the discovery of many more recombination genes, the phenomenon of post replication-recombination repair, the invention of the SOS hypothesis and the discovery of genes encoding proteins with similar primary structure and function in all major groups of organisms (Clark, 1996).
Germline specific expression and the sterility of DMC1-deficient mice defined DMC1 as a meiosis-specific component of the homologous recombination complex (Habu, 1996; Pittman, 1998). However, genome-wide search failed to identify a clear Dmc1 homolog in Drosophila. Considering that a Dmc1-like gene would be expressed exclusively in the germline, whether DmRad51/SpnA or any of the other Drosophila Rad51 family members are meiosis specific was examined by determining germline and soma gene expression of the five Drosophila Rad51 genes by RT-PCR. Rad51 gene expression profiles from wild-type females, flies from tudor mutant females, which lack germline, and to males, which fail to undergo meiotic recombination, were compared. For each Rad51 gene, primers were designed to specifically amplify a fragment of the corresponding transcript. As a control for germline-specific expression, oskar and nanos RNA were examined by RT-PCR. spnA, spnB and rad51C RNA are expressed in both males and females. Furthermore, their expression is not limited to the female germline. Interestingly, rad51D and spnD RNA appear to be expressed almost exclusively in the germline of adult females. Thus, Drosophila may have two Rad51 family members that are specifically involved in meiotic recombination and functionally equivalent to Dmc1 (Staeva-Vieira, 2003).
To clarify RAD51 interactions controlling homologous recombination, the crystal structure of the full-length RAD51 homolog from Pyrococcus furiosus is reported. The structure reveals how RAD51 proteins assemble into inactive heptameric rings and active DNA-bound filaments matching three-dimensional electron microscopy reconstructions. A polymerization motif (RAD51-PM) tethers individual subunits together to form assemblies. Subunit interactions support an allosteric 'switch' promoting ATPase activity and DNA binding roles for the N-terminal domain helix-hairpin-helix (HhH) motif. Structural and mutational results characterize RAD51 interactions with the breast cancer susceptibility protein BRCA2 in higher eukaryotes. A designed P. furiosus RAD51 mutant binds BRC repeats and forms BRCA2-dependent nuclear foci in human cells in response to gamma-irradiation-induced DNA damage, similar to human RAD51. These results show that BRCA2 repeats mimic the RAD51-PM and imply analogous RAD51 interactions with RAD52 and RAD54. Both BRCA2 and RAD54 may act as antagonists and chaperones for RAD51 filament assembly by coupling RAD51 interface exchanges with DNA binding. Together, these structural and mutational results support an interface exchange hypothesis for coordinated protein interactions in homologous recombination (Shin, 2003).
The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. The rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, Rad51 protein is found to be similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae (Shinohara, 1992).
The RAD54 and RAD51 genes are involved in genetic recombination and double-strand break repair in the yeast Saccharomyces cerevisiae. The Rad51 protein is thought to be a yeast analogue of the Eschericia coli recA gene product and catalyzes strand exchange between homologous single- and double-stranded DNAs in vitro. RAD54 exhibits homologies to several known ATPases and is a member of the SWI2/MOT1 family. The Rad54 protein interacts with the Rad51 protein both in vivo and in vitro, and the NH2-terminal 115 residues of the Rad54 protein are shown to be necessary for this interaction. These data imply that the Rad54 protein is part of a multiprotein yeast recombination complex (Jiang, 1996).
Rad51p is a eukaryotic homolog of RecA, the central homologous pairing and strand exchange protein in Escherichia coli. Rad54p belongs to the Swi2p/Snf2p family of DNA-stimulated ATPases. Both proteins are also important members of the RAD52 group, which controls recombinational DNA damage repair of double-strand breaks and other DNA lesions in Saccharomyces cerevisiae. Rad51 and Rad54 proteins are shown by genetic, molecular and biochemical criteria to interact. Strikingly, overexpression of Rad54p can functionally suppress the UV and methyl methanesulfonate sensitivity caused by a deletion of the RAD51 gene. However, no suppression is observed for the defects of rad51 cells in the repair of gamma-ray-induced DNA damage, mating type switching or spontaneous hetero-allelic recombination. This suppression is genetically dependent on the presence of two other members of the recombinational repair group, RAD55 and RAD57. These data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair. Since both proteins are conserved throughout evolution from yeasts to humans, a similar protein-protein interaction may be expected in other organisms (Clever, 1997).
The Schizosaccharomyces pombe rhp51+, rad22+ and rhp54+ genes are homologous to RAD51, RAD52 and RAD54 respectively, which are indispensable in the recombinational repair of double-strand breaks (DSBs) in Saccharomyces cerevisiae. The rhp51Delta and rhp54Delta strains are extremely sensitive to ionizing radiation; the rad22Delta mutant turns out to be much less sensitive. Homologous recombination in these mutants was studied by targeted integration at the leu1-32 locus. These experiments revealed that rhp51Delta and rhp54Delta are equally impaired in the integration of plasmid molecules (15-fold reduction), while integration in the rad22Delta mutant is only reduced by a factor of two. Blot-analysis demonstrates that the majority of the leu+ transformants of the wild-type and rad22Delta strains have integrated one or more copies of the vector. Gene conversion events are observed in less than 10% of the transformants. Interestingly, the relative contribution of gene conversion events is much higher in a rhp51Delta and a rhp54Delta background. Meiotic recombination is hardly affected in the rad22Delta mutant. The rhp51Delta and rhp54Delta strains also show minor deficiencies in this type of recombination. The viability of spores is 46% in the rad22Delta strain and 27% in the rhp54Delta strain, as compared with wild-type cells. However, in the rhp51Delta mutant the spore viability is only 1.7%, suggesting an essential role for Rhp51 in meiosis. The function of Rhp51 and Rhp54 in damage repair and recombination resembles the role of Rad51 and Rad54 in S. cerevisiae. Compared with Rad52 from S. cerevisiae, Rad22 has a much less prominent role in the recombinational repair pathway in S. pombe (Muris, 1997).
The generation of a double-strand break in the Saccharomyces cerevisiae genome is a potentially catastrophic event that can induce cell-cycle arrest or ultimately result in loss of cell viability. The repair of such lesions is strongly dependent on proteins encoded by the RAD52 epistasis group of genes (RAD50-55, RAD57, MRE11, XRS2), as well as the RFA1 and RAD59 genes. rad52 mutants exhibit the most severe phenotypic defects in double-strand break repair, but almost nothing is known about the biochemical role of Rad52 protein. Rad51 protein promotes DNA strand exchange and acts similarly to RecA protein. Yeast Rad52 protein interacts with Rad51 protein, binds single-stranded DNA and stimulates annealing of complementary single-stranded DNA. Rad52 protein is found to stimulate DNA strand exchange by targeting Rad51 protein to a complex of replication protein A (RPA) with single-stranded DNA. Rad52 protein affects an early step in the reaction, presynaptic filament formation, by overcoming the inhibitory effects of the competitor, RPA. Furthermore, stimulation is dependent on the concerted action of both Rad51 protein and RPA, implying that specific protein-protein interactions between Rad52 protein, Rad51 protein and RPA are required (New, 1998).
The Saccharomyces cerevisiae RAD51 and RAD54 genes are both required for the occurrence of homologous recombination and for the repair of double-stranded DNA breaks. Previous studies have indicated that Rad51 protein, together with the single-stranded DNA-binding factor replication protein A (RPA), can promote the formation of heteroduplex DNA, which is a key intermediate in homologous recombination. Rad54 protein has now been purified to near homogeneity and its molecular function has been biochemically tested. Rad54 protein possesses a double-stranded DNA-dependent ATPase activity, and it interacts with the Rad51 protein. Addition of Rad54 protein to reactions containing Rad51 strongly stimulates the rate of pairing between homologous single-stranded and double-stranded DNA molecules. It is concluded that Rad54 acts to overcome kinetic impediments that would limit homologous DNA pairing between recombining chromosomes in vivo (Petukhova, 1998).
Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key intermediate in recombination processes. Rad54 is monomeric in solution, but forms a dimer/oligomer on DNA. Rad54 dimer/oligomer alters the conformation of the DNA double helix in an ATP-dependent manner, as revealed by a change in the DNA linking number in a topoisomerase I-linked reaction. DNA conformational alteration does not occur in the presence of non-hydrolyzable ATP analogues, nor when mutant rad54 proteins defective in ATP hydrolysis replace Rad54. Accordingly, the Rad54 ATPase activity is shown to be required for biological function in vivo and for promoting Rad51-mediated homologous DNA pairing in vitro. Taken together, the results are consistent with a model in which a Rad54 dimer/oligomer promotes nascent heteroduplex joint formation via a specific interaction with Rad51 protein and an ability to transiently unwind duplex DNA (Petukhova, 1999).
In Saccharomyces cerevisiae, the RAD51 and RAD52 genes are involved in recombination and in repair of damaged DNA. The RAD51 gene is a structural and functional homolog of the recA gene; the gene product participates in strand exchange and single-stranded-DNA-dependent ATP hydrolysis by means of nucleoprotein filament formation. The RAD52 gene is important in RAD51-mediated recombination. Binding of this protein to Rad51 suggests that they cooperate in recombination. Homologs of both Rad51 and Rad52 are conserved from yeast to humans, suggesting that the mechanisms used for pairing homologous DNA molecules during recombination may be universal in eukaryotes. Rad52 protein is shown to stimulate Rad51 reactions and binding to Rad51 is necessary for this stimulatory effect. It is concluded that this binding is crucial in recombination and that it facilitates the formation of Rad51 nucleoprotein filaments (Shinohara, 1998).
Yeast Rad51 recombinase has only minimal ability to form D loop. Addition of Rad54 renders D loop formation by Rad51 efficient, even when topologically relaxed DNA is used as substrate. Treatment of the nucleoprotein complex of Rad54 and relaxed DNA with topoisomerases reveals dynamic DNA remodeling to generate unconstrained negative and positive supercoils. DNA remodeling requires ATP hydrolysis by Rad54 and is stimulated by Rad51-DNA nucleoprotein complex. A marked sensitivity of DNA undergoing remodeling to P1 nuclease indicates that the negative supercoils produced lead to transient DNA strand separation. Thus, a specific interaction of Rad54 with the Rad51-ssDNA complex enhances the ability of the former to remodel DNA and allows the latter to harvest the negative supercoils generated for DNA joint formation (Van Komen, 2000).
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 carries out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibits dsDNA-specific ATPase activity. Rad54 protein stimulates Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation is accompanied by a concurrent increase in the formation of heteroduplex DNA. These 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 does not stimulate DNA strand exchange by increasing presynaptic complex formation. It is concluded that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments (Solinger, 2001a).
Rad54 and Rad51 are important proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. Rad51 protein forms nucleoprotein filaments on single-stranded DNA, and Rad54 protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Saccharomyces cerevisiae Rad54 protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depends on the ATPase activity of Rad54 protein and on specific protein:protein interactions between the yeast Rad54 and Rad51 proteins (Solinger, 2001b).
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 (Fortin, 2002).
Rad54 protein is a Snf2-like ATPase with a specialized function in the recombinational repair of DNA damage. Rad54 is thought to stimulate the search of homology via formation of a specific complex with the presynaptic Rad51 filament on single-stranded DNA. This study addresses the interaction of Rad54 with Rad51 filaments on double-stranded (ds) DNA, an intermediate in DNA strand exchange with unclear functional significance. Saccharomyces cerevisiae Rad54 is shown to exert distinct modes of ATPase activity on partially and fully saturated filaments of Rad51 protein on dsDNA. The highest ATPase activity is observed on dsDNA containing short patches of yeast Rad51 filaments resulting in a 6-fold increase compared with protein-free DNA. This enhanced ATPase mode of yeast Rad54 can also be elicited by partial filaments of human Rad51 protein but to a lesser extent. In contrast, the interaction of Rad54 protein with duplex DNA fully covered with Rad51 is entirely species-specific. When yeast Rad51 fully covers dsDNA, Rad54 protein hydrolyzes ATP in a reduced mode at 60%-80% of its rate on protein-free DNA. Instead, saturated filaments with human Rad51 fail to support the yeast Rad54 ATPase. It is suggested that the interaction of Rad54 with dsDNA-Rad51 complexes is of functional importance in homologous recombination (Kiianitsa, 2002).
Saccharomyces cerevisiae Rad51, Rad54, and replication protein A (RPA) proteins work in concert to make heteroduplex DNA joints during homologous recombination. With plasmid length DNA substrates, maximal DNA joint formation is observed with amounts of Rad51 substantially below what is needed to saturate the initiating single-stranded DNA template, and, relative to Rad51, Rad54 is needed in only catalytic quantities. RPA is still indispensable for optimal reaction efficiency, but its role in this instance is to sequester free single-stranded DNA, which otherwise inhibits Rad51 and Rad54 functions. Rad54 helps overcome various reaction constraints in DNA joint formation. These results thus shed light on the function of Rad54 in the Rad51-mediated homologous DNA pairing reaction and also reveal a novel role of RPA in the presynaptic stage of this reaction (Van Komen, 2002).
Repairing a double-strand break by homologous recombination requires binding of the strand exchange protein Rad51p to ssDNA, followed by synapsis with a homologous donor. Chromatin immunoprecipitation has been used to monitor the in vivo association of Saccharomyces cerevisiae Rad51p with both the cleaved MATa locus and the HMLα donor. Localization of Rad51p to MAT precedes its association with HML, providing evidence of the time needed for the Rad51 filament to search the genome for a homologous sequence. Rad51p binding to ssDNA requires Rad52p. The absence of Rad55p delays Rad51p binding to ssDNA and prevents strand invasion and localization of Rad51p to HMLα. Lack of Rad54p does not significantly impair Rad51p recruitment to MAT or its initial association with HMLα; however, Rad54p is required at or before the initiation of DNA synthesis after synapsis has occurred at the 3' end of the invading strand (Sugawara, 2003).
Repair of a double-strand break (DSB) often occurs by gene conversion, a homologous recombination event. Homologous recombination depends on a search for homology by the ends of the broken chromosome to locate an intact donor sequence that could be used as a template for DNA repair. In budding yeast and other eukaryotes, the search for homology is facilitated by the DNA strand exchange protein, Rad51p, the homolog of the bacterial RecA protein. In vitro, Rad51p, like RecA, forms extensive Rad51p-DNA filaments, with 3 bp bound per monomer. The RecA or Rad51p filament can catalyze strand exchange between ssDNA and a homologous dsDNA. This process is greatly facilitated by prior exposure of the ssDNA to a ssDNA binding protein, SSB in bacteria, or RPA in eukaryotes. These ssDNA binding proteins appear to facilitate the polymerization of RecA/Rad51p across ssDNA regions that can form secondary structure (Sugawara, 2003 and references therein).
Incubation of RPA with ssDNA before the addition of Rad51p, however, reduces formation of Rad51 filaments. Either Rad52p or the Rad55p/Rad57p heterodimer can overcome this inhibition and mediate the loading of Rad51p onto ssDNA. Rad54p shares sequence similarity with the Swi2/Snf2 family of chromatin-remodeling proteins and appears to act at a later step in strand exchange, to extend heteroduplex DNA, and to alter DNA conformation during the synaptic and/or postsynaptic phases of strand exchange. In vivo, Rad51p-mediated recombination in S. cerevisiae requires the participation of Rad52p, Rad54p, Rad55p, and Rad57p. Deletions of RAD52 are the most defective in spontaneous and DSB-induced recombination, since this protein is required both for Rad51p-mediated and Rad59p-mediated recombination events. The requirements for Rad55p and Rad57p are the least stringent, as defects are often seen only at low temperature. This defect can be suppressed by the overexpression of Rad51p (Sugawara, 2003 and references therein).
One of the best-studied homologous recombination events is the HO endonuclease-induced switching of the MATa locus, using HMLα as the donor template during gene conversion. A galactose-inducible GAL::HO gene provides the means to induce the DSB synchronously in all cells of the population. Physical monitoring of MAT DNA has shown that the DSB ends are first resected by 5' to 3' exonucleases, presumably so that the 3'-ended ssDNA can recruit the Rad51p strand exchange protein and undergo strand invasion of the donor and initiate new DNA synthesis. Although the formation of a strand invasion intermediate has not been directly demonstrated, the next step in the process, the use of the 3' end of the invading strand as a primer to copy the template DNA, has been detected by PCR. This was observed to occur about 30 min after the appearance of the DSB and 30 min before the recombination event was completed. HO cleavage and 5' to 3' resection are normal in mutant cells lacking the auxiliary recombination proteins Rad52p, Rad54p, Rad55p, and Rad57p, but the primer extension step does not occur; however, in some strain backgrounds there is a small amount of MAT switching in rad54Δ mutants (Sugawara, 2003 and references therein).
To determine more precisely the recombination steps that are prevented in the absence of the auxiliary proteins, chromatin immunoprecipitation (ChIP) was used to follow the ability of Rad51p to associate with ssDNA formed at MAT and with DNA sequences at HML as well as a model for the synapsis of MAT and HML during gene conversion. This approach has enabled distinct functions for Rad52p, Rad54p, and Radd55p/Rad57p to be seen in the Rad51p-mediated process. It is concluded that Rad52p is required for the recruitment of Rad51p to a DSB, whereas the rate of recruitment is reduced or delayed in the absence of Rad55p/Rad57p. At the step of strand exchange, Rad55p/Rad57p is required for recruitment of Rad51p to the donor whereas Rad54p is not, suggesting that synaptic association between MAT and the HML donor does not need Rad54p. Rad54p is required at a postsynaptic step to enable the completion of DNA repair (Sugawara, 2003).
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. 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, it is proposed that Rad54 facilitates chromatin remodeling and, perhaps more generally, protein clearing at the homology search step of genetic recombination (Alexeev, 2003).
Saccharomyces cells with a single unrepaired double-strand break adapt after checkpoint-mediated G(2)/M arrest. Rad51 and Rad52 recombination proteins play key roles in adaptation. Cells lacking Rad51p fail to adapt, but deleting RAD52 suppresses rad51Delta. rad52Delta also suppresses adaptation defects of srs2Delta mutants but not those of yku70Delta or tid1Delta mutants. Neither rad54Delta nor rad55Delta affects adaptation. A Rad51 mutant that fails to interact with Rad52p is adaptation defective; conversely, a C-terminal truncation mutant of Rad52p, impaired in interaction with Rad51p, is also adaptation defective. In contrast, rad51-K191A, a mutation that abolishes recombination and results in a protein that does not bind to single-stranded DNA (ssDNA), supports adaptation, as do Rad51 mutants impaired in interaction with Rad54p or Rad55p. An rfa1-t11 mutation in the ssDNA binding complex RPA partially restores adaptation in rad51Delta mutants and fully restores adaptation in yku70Delta and tid1Delta mutants. Surprisingly, although neither rfa1-t11 nor rad52Delta mutants are adaptation defective, the rad52Delta rfa1-t11 double mutant fails to adapt and exhibits the persistent hyperphosphorylation of the DNA damage checkpoint protein Rad53 after HO induction. It is suggested that monitoring of the extent of DNA damage depends on independent binding of RPA and Rad52p to ssDNA, with Rad52p's activity modulated by Rad51p whereas RPA's action depends on Tid1p (Lee, 2003).
To prevent genome instability, recombination between sequences that contain mismatches (homeologous recombination) is suppressed by the mismatch repair (MMR) pathway. To understand the interactions necessary for this regulation, the genetic requirements for the inhibition of homeologous recombination were examined using mutants in the RAD52 epistasis group of Saccharomyces cerevisiae. The use of a chromosomal inverted-repeat recombination assay to measure spontaneous recombination between 91% and 100% identical sequences demonstrates differences in the fidelity of recombination in pathways defined by their dependence on RAD51 and RAD59. In addition, the regulation of homeologous recombination in rad51 and rad59 mutants displays distinct patterns of inhibition by different members of the MMR pathway. Whereas the requirements for the MutS homolog, MSH2, and the MutL homolog, MLH1, in the suppression of homeologous recombination are similar in rad51 strains, the loss of MSH2 causes a greater loss in homeologous recombination suppression than does the loss of MLH1 in a rad59 strain. The nonequivalence of the regulatory patterns in the wild-type and mutant strains suggests an overlap between the roles of the RAD51 and RAD59 gene products in potential cooperative recombination mechanisms used in wild-type cells (Spell, 2003).
A chromosome fragmentation assay was used to measure the efficiency and genetic control of break-induced replication (BIR) in Saccharomyces cerevisiae. Formation of a chromosome fragment by de novo telomere generation at one end of the linear vector and recombination-dependent replication of 100 kb of chromosomal sequences at the other end of the vector occurred at high frequency in wild-type strains. RAD51 was required for more than 95% of BIR events involving a single-end invasion and was essential when two BIR events were required for generation of a chromosome fragment. The similar genetic requirements for BIR and gene conversion suggest a common strand invasion intermediate in these two recombinational repair processes. Mutation of RAD50 or RAD59 conferred no significant defect in BIR in either RAD51 or rad51 strains. RAD52 is essential for BIR at unique chromosomal sequences, although rare recombination events were detected between the subtelomeric Y' repeats (Davis, 2004).
The Saccharomyces cerevisiae RDH54-encoded product, a member of the Swi2/Snf2 protein family, is needed for mitotic and meiotic interhomologue recombination and DNA repair. Previous biochemical studies employing Rdh54 purified from yeast cells have shown DNA-dependent ATP hydrolysis and DNA supercoiling by this protein, indicative of a DNA translocase function. Importantly, Rdh54 physically interacts with the Rad51 recombinase and promotes D-loop formation by the latter. Unfortunately, the low yield of Rdh54 from the yeast expression system has greatly hampered the progress on defining the functional interactions of this Swi2/Snf2-like factor with Rad51. This study describes an E. coli expression system and purification scheme that together provide milligram quantities of nearly homogeneous Rdh54. Using this material, it has been demonstrated that Rdh54-mediated DNA supercoiling leads to transient DNA strand opening. Furthermore, at the expense of ATP hydrolysis, Rdh54 removes Rad51 from DNA. Evidence is presented that the Rad51 binding domain resides within the N terminus of Rdh54. Accordingly, N-terminal truncation mutants of Rdh54 that fail to bind Rad51 are also impaired for functional interactions with the latter. Interestingly, the rdh54 K352R mutation that ablates ATPase activity engenders a DNA repair defect even more severe than that seen in the rdh54Delta mutant. These results provide molecular information concerning the role of Rdh54 in homologous recombination and DNA repair, and they also demonstrate the functional significance of Rdh54.Rad51 complex formation. The Rdh54 expression and purification procedures described in this study should facilitate the functional dissection of this DNA recombination/repair factor (Chi, 2006).
Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Evidence is provided that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, a class of rad52 mutants is described that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of these conclusions, it is shown that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells (Lettier, 2006; full text of article).
Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, in vivo functions were examined of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Δ mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Δ mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Δ and rhp57Δ mutants and completely abolished in an swi5Δ rhp57Δ double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator (Akamatsu, 2007).
Meiotic recombination-related DNA synthesis (MRDS) was analyzed in Saccharomyces cerevisiae by specifically timed incorporation of thymidine analogs into chromosomes. Lengths and positions of incorporation tracts were determined relative to a known recombination hot spot along DNA, as was the timing and localization of incorporation relative to forming and formed synaptonemal complex in spread chromosomes. Distinct patterns could be specifically associated with the majority cross-over and non-cross-over recombination processes. The results obtained provide direct evidence for key aspects of current consensus recombination models, provide information regarding temporal and spatial relationships between non-cross-over formation and the synaptonemal complex, and raise the possibility that removal of RecA homolog Rad51 plays a key role in regulating onset of MRDS. Finally, classical observations on MRDS in Drosophila, mouse, and lily are readily mapped onto the findings presented in this study, providing further evidence for a broadly conserved meiotic recombination process (Terasawa, 2007).
Heterochromatin plays a key role in protection of chromosome integrity by suppressing homologous recombination. In Saccharomyces cerevisiae, Sir2p, Sir3p, and Sir4p are structural components of heterochromatin found at telomeres and the silent mating-type loci. This study investigated whether incorporation of Sir proteins into minichromosomes regulates early steps of recombinational repair in vitro. It was found that addition of Sir3p to a nucleosomal substrate is sufficient to eliminate yRad51p-catalyzed formation of joints, and that this repression is enhanced by Sir2p/Sir4p. Importantly, Sir-mediated repression requires histone residues that are critical for silencing in vivo. Moreover, the SWI/SNF chromatin-remodeling enzyme facilitates joint formation by evicting Sir3p, thereby promoting subsequent Rad54p-dependent formation of a strand invasion product. These results suggest that recombinational repair in the context of heterochromatin presents additional constraints that can be overcome by ATP-dependent chromatin-remodeling enzymes (Sinha, 2009).
Saccharomyces cerevisiae Rad51 protein is the paradigm for eukaryotic ATP-dependent DNA strand exchange proteins. To explain some of the unique characteristics of DNA strand exchange promoted by Rad51 protein, when compared with its prokaryotic homolog the Escherichia coli RecA protein, the DNA binding properties of the Rad51 protein were analyzed. Rad51 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP- and Mg2+-dependent manner, over a wide range of pH, with an apparent binding stoichiometry of approximately 1 protein monomer per 4 (+/-1) nucleotides or base pairs, respectively. Only dATP and adenosine 5'-gamma-(thiotriphosphate) (ATPgammaS) can substitute for ATP, but binding in the presence of ATPgammaS requires more than a 5-fold stoichiometric excess of protein. Without nucleotide cofactor, Rad51 protein binds both ssDNA and dsDNA but only at pH values lower than 6.8; in this case, the apparent binding stoichiometry covers the range of 1 protein monomer per 6-9 nucleotides or base pairs. Therefore, Rad51 protein displays two distinct modes of DNA binding. These binding modes are not inter-convertible; however, their initial selection is governed by ATP binding. On the basis of these DNA binding properties, it is concluded that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during presynapsis, Rad51 protein interacts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoprotein filament; during synapsis, Rad51 protein inactivates the homologous dsDNA partner by directly binding to it (Zaitseva, 1999).
Crossover and noncrossover recombinants can form by two different pathways during meiotic recombination in Saccharomyces cerevisiae. The MER3 gene is known to affect selectively crossover, but not noncrossover, recombination. The Mer3 protein is a DNA helicase that unwinds duplex DNA in the 3' to 5' direction. To define the underlying molecular steps of meiotic recombination, the role of Mer3 helicase in DNA strand exchange promoted by Rad51 protein was investigated. It was found that Mer3 helicase does not function as an initiator of DNA pairing events but, rather, it stimulates DNA heteroduplex extension in the 3' -> 5' direction, relative to the incoming (or displaced) single-stranded DNA. Conversely, Mer3 helicase blocks DNA heteroduplex extension in the 5' -> 3' direction. These results support the idea that Mer3 helicase stabilizes nascent joint molecules via DNA heteroduplex extension to permit capture of the second processed end of a double-stranded DNA break, a step that is required for crossover recombinant product formation (Mazina, 2004).
The meiosis-specific recombinase Dmc1 plays a critical role in DNA strand exchange in budding yeast. Tid1/Rdh54, a member of the Swi2/Snf2 family of DNA translocases, has been shown to stimulate Dmc1-dependent recombination. Tid1 and its budding yeast paralog Rad54 have a variety of biochemical activities that may contribute to their biological function. This study demonstrates that Dmc1 can associate with chromatin in the absence of DNA double-strand breaks (DSBs), and Tid1 suppresses this association. Chromatin immunoprecipitation experiments indicate that an activity shared by Tid1 and Rad54 is required for normal assembly of Dmc1 at DSB sites in preparation for recombination. These results lead to a model in which the ATP hydrolysis-dependent DNA translocase activity of Tid1 acts to promote dissociation of Dmc1 from nonreombinogenic sites on chromatin, with Rad54 being able to substitute for this function in the absence of Tid1. The tendency of Dmc1 to form unproductive interactions with chromatin is proposed to be a consequence of the mechanism of strand exchange. The results raise the possibility that ATP hydrolysis-dependent disruption of nonproductive recombinase-DNA interactions is a feature shared with other homologous recombination systems (Holzen 2006).
DMC1 is a new meiosis-specific yeast gene. Dmc1 protein is structurally similar to bacterial RecA proteins. dmc1 mutants are defective in reciprocal recombination, accumulate double-strand break (DSB) recombination intermediates, fail to form normal synaptonemal complex (SC), and arrest late in meiotic prophase. dmc1 phenotypes are consistent with a functional relationship between Dmc1 and RecA, and thus eukaryotic and prokaryotic mechanisms for homology recognition and strand exchange may be related. dmc1 phenotypes provide further evidence that recombination and SC formation are interrelated processes and are consistent with a requirement for DNA-DNA interactions during SC formation. dmc1 mutations confer prophase arrest. Additional evidence suggests that arrest occurs at a meiosis-specific cell cycle 'checkpoint' in response to a primary defect in prophase chromosome metabolism. DMC1 is homologous to yeast's RAD51 gene, supporting the view that mitotic DSB repair has been recruited for use in meiotic chromosome metabolism (Bishop, 1992).
DMC1, the meiosis-specific eukaryotic homolog of bacterial recA, is required for completion of meiotic recombination and cell cycle progression past prophase. In a dmc1 mutant, double strand break recombination intermediates accumulate and cells arrest in prophase. Genes have been isolated which, when present at high copy numbers, suppress the meiotic arrest phenotype conferred by dmc1 mutations. Among the genes isolated were two which suppress arrest by altering the recombination process. REC114 suppresses formation of double strand break (DSB) recombination intermediates. The low viability of spores produced by dmc1 mutants carrying high copy numbers of REC114 is rescued when reductional segregation is bypassed by mutation of spo13. High copy numbers of RAD54 suppress dmc1 arrest, promote DSB repair, and allow formation of viable spores following reductional segregation. Analysis of the combined effects of a null mutation in RED1, a gene required for meiotic chromosome structure, with null mutations in RAD54 and DMC1 shows that RAD54, while not normally important for repair of DSBs during meiosis, is required for efficient repair of breaks by the intersister recombination pathway that operates in red1 dmc1 double mutants. It is concluded that over-expression of REC114 suppresses meiotic arrest by preventing formation of DSBs. High copy numbers of RAD54 activate a DMC1-independent mechanism that promotes repair of DSBs by homology-mediated recombination. The ability of RAD54 to promote DMC1-independent recombination is proposed to involve suppression of a constraint that normally promotes recombination between homologous chromatids rather than sisters (Bishop, 1999).
Dmc1 and Rad51 are eukaryotic RecA homologs that are involved in meiotic recombination. The expression of Dmc1 is limited to meiosis, whereas Rad51 is expressed in mitosis and meiosis. Dmc1 and Rad51 have unique and overlapping functions during meiotic recombination. Dmc1 protein has been purified from the budding yeast Saccharomyces cerevisiae and its biochemical activity has been characterized. The protein has a weak DNA-dependent ATPase activity and binds both single-strand DNA (ssDNA) and double-strand DNA. Electrophoretic mobility shift assays suggest that DNA binding by Dmc1 is cooperative. Dmc1 renatures linearized plasmid DNA with first order reaction kinetics and without requiring added nucleotide cofactor. In addition, Dmc1 catalyzes strand assimilation of ssDNA oligonucleotides into homologous supercoiled duplex DNA in a reaction promoted by ATP or the non-hydrolyzable ATP analogue AMP-PNP (Hong, 2001).
Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. Budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases-Srs2 and Sgs1-that also attenuate crossing over. By chromatin immunoprecipitation, targeting of Mph1 to double-strand breaks was found in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from this analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks (Prakash, 2009).
Since Mph1 influences the outcome rather than the efficiency of recombinational repair events, it very likely acts by shunting a DNA intermediate into the non-crossover generating synthesis-dependent strand annealing pathway. As revealed in ChIP experiments, Mph1 is targeted to DSBs in cells, suggesting that its action in recombination regulation is direct. Biochemical results provided evidence that Mph1 regulates recombination pathway choice by dissociating the invading DNA strand from the Rad51-made D-loop (Prakash, 2009).
XRCC3 is a RAD51 paralog that functions in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). XRCC3 mutation causes severe chromosome instability. XRCC3 mutant cells display radically altered HR product spectra, with increased gene conversion tract lengths, increased frequencies of discontinuous tracts, and frequent local rearrangements associated with HR. These results indicate that XRCC3 function is not limited to HR initiation, but extends to later stages in formation and resolution of HR intermediates, possibly by stabilizing heteroduplex DNA. The results further demonstrate that HR defects can promote genomic instability not only through failure to initiate HR (leading to nonhomologous repair) but also through aberrant processing of HR intermediates. Both mechanisms may contribute to carcinogenesis in HR-deficient cells (Brennaman, 2002).
Homologous recombinational repair preserves chromosomal integrity by removing double-strand breaks, crosslinks, and other DNA damage. In eukaryotic cells, the Rad51 paralogs (XRCC2/3, Rad51B/C/D) are involved in this process, although their exact functions are largely undetermined. All five paralogs contain ATPase motifs, and XRCC3 exists in a single complex with Rad51C. To examine the function of this Rad51C-XRCC3 complex, mammalian expression vectors were generated that produce human wild-type XRCC3 or mutant XRCC3 with either a non-conservative mutation (K113A) or a conservative mutation (K113R) in the GKT Walker A box of the ATPase motif. The three vectors were independently transfected into Xrcc3-deficient irs1SF CHO cells. Wild-type XRCC3 complemented irs1SF cells, albeit to varying degrees, while ATPase mutants had no complementing activity, even when the mutant protein was expressed at comparable levels to those in wild-type-complemented clones. Because of the mutants dysfunction, it is proposed that ATP binding and hydrolyzing activities of XRCC3 are essential. In vitro complex formation by wild-type and mutant XRCC3 with His6-tagged Rad51C was tested upon co-expression in bacteria, nickel affinity purification, and western blotting. Wild-type and K113A mutant XRCC3 formed stable complexes with Rad51C and co-purified with Rad51C, while the K113R mutant did not and was predominantly insoluble. Addition of 5 mM ATP, but not ADP, also abolished complex formation by the wild-type proteins. These results suggest that XRCC3 is likely to regulate the dissociation and formation of Rad51C-XRCC3 complex through ATP binding and hydrolysis, with both processes being essential for the complex's ability to participate in homologous recombination repair (Yamada, 2004).
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. 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) exhibit 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 results in telomere erosion and chromosome fusion. It is concluded that RAD51D plays a dual cellular role in both the repair of DNA double-strand breaks and telomere protection against attrition and fusion (Tarsounas, 2004).
Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment. This study clarifies the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. These findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing (Krejci, 2003).
Homologous recombination is a ubiquitous process with key functions in meiotic and vegetative cells for the repair of DNA breaks. It is initiated by the formation of single-stranded DNA on which recombination proteins bind to form a nucleoprotein filament that is active in searching for homology, in the formation of joint molecules and in the exchange of DNA strands. This process contributes to genome stability but it is also potentially dangerous to cells if intermediates are formed that cannot be processed normally and thus are toxic or generate genomic rearrangements. Cells must therefore have developed strategies to survey recombination and to prevent the occurrence of such deleterious events. In Saccharomyces cerevisiae, genetic data have shown that the Srs2 helicase negatively modulates recombination, and later experiments suggested that it reverses intermediate recombination structures. DNA strand exchange mediated in vitro by Rad51 is inhibited by Srs2, and Srs2 disrupts Rad51 filaments formed on single-stranded DNA. These data provide an explanation for the anti-recombinogenic role of Srs2 in vivo and highlight a previously unknown mechanism for recombination control (Veaute, 2003).
S-phase cells overcome chromosome lesions through replication-coupled recombination processes that seem to be assisted by recombination-dependent DNA structures and/or replication-related sister chromatid junctions. RecQ helicases, including yeast Sgs1 and human BLM, have been implicated in both replication and recombination and protect genome integrity by preventing unscheduled mitotic recombination events. The RecQ helicase-mediated mechanisms controlling genome stability by analyzing replication forks encountering a damaged template were examined in sgs1 cells. In sgs1 mutants, recombination-dependent cruciform structures accumulate at damaged forks. Their accumulation requires Rad51 protein; this is counteracted by Srs2 DNA helicase, and does not prevent fork movement. Sgs1, but not Srs2, promotes resolution of these recombination intermediates. A functional Rad53 checkpoint kinase that is known to protect the integrity of the sister chromatid junctions is required for the accumulation of recombination intermediates in sgs1 mutants. Finally, top3 and top3 sgs1 mutants accumulate the same structures as sgs1 cells. It is suggested that, in sgs1 cells, the unscheduled accumulation of Rad51-dependent cruciform structures at damaged forks result from defective maturation of recombination-dependent intermediates that originate from the replication-related sister chromatid junctions. These findings might contribute to explaining some of the recombination defects of BLM cells (Liberi, 2005).
In this study, cell cycle-dependent expression of human and rodent RAD51 and RAD52 proteins was monitored using two approaches. (1) Flow cytometric measurements of DNA content and immunofluorescence were used to determine the phase-specific levels of RAD51 and RAD52 protein expression in irradiated and control populations. The expression of both proteins is lowest in G0/G1, increases in S and reaches a maximum in G2/M. No difference is found in the whole-cell level of RAD51 or RAD52 protein expression between gamma-irradiated and control cell populations. (2) Cell cycle-dependent protein expression was confirmed by Western analysis of populations synchronized in G0, G1 and G2 phases. Analysis of V3, a hamster equivalent of SCID, indicates that the protein level increases of RAD51 and RAD52 from G0 to G1/S/G2 do not require DNA-PK (Chen, 1997).
Rad51, a eukaryotic RecA homolog, plays a central role in homologous recombinational repair of DNA double-strand breaks (DSBs) in yeast and is conserved from yeast to human. Rad51 shows punctuate nuclear localization in human cells, called Rad51 foci, typically during the S phase. However, the topological relationships that exist in human S phase nuclei between Rad51 foci and damaged chromatin have not been studied thus far. This study reports the results of ultraviolet microirradiation experiments of small nuclear areas and on whole cell ultraviolet C (UVC) irradiation experiments performed with a human fibroblast cell line. Before UV irradiation, nuclear DNA was sensitized by the incorporation of halogenated thymidine analogues. These experiments demonstrate the redistribution of Rad51 to the selectively damaged, labeled chromatin. Rad51 recruitment takes place from Rad51 foci scattered throughout the nucleus of nonirradiated cells in S phase. The preferential association of Rad51 foci with postreplicative chromatin in contrast to replicating chromatin is demonstrated using a double labeling procedure with halogenated thymidine analogues. This finding supports a role of Rad51 in recombinational repair processes of DNA damage present in postreplicative chromatin (Tashiro, 2000).
The receptor for insulin-like growth factor I (IGF-IR) controls normal and pathological growth of cells. DNA repair pathways represent an unexplored target through which the IGF-IR signaling system might support pathological growth leading to cellular transformation. However, this study demonstrates that IGF-I stimulation supports homologous recombination-directed DNA repair (HRR). This effect involves an interaction between Rad51 and the major IGF-IR signaling molecule, insulin receptor substrate 1 (IRS-1). The binding occurs within the cytoplasm, engages the N-terminal domain of IRS-1, and is attenuated by IGF-I-mediated IRS-1 tyrosine phosphorylation. In the absence of IGF-I stimulation, or if mutated IGF-IR fails to phosphorylate IRS-1, localization of Rad51 to the sites of damaged DNA is diminished. These results point to a direct role of IRS-1 in HRR and suggest a novel role for the IGF-IR/IRS-1 axis in supporting the stability of the genome (Trojanek, 2003).
The human testis Rad51 protein, a structural homolog of E. coli RecA, binds single- and double-stranded DNA and exhibits DNA-dependent ATPase activity. Using circular ssDNA and linear dsDNA (3.0 kb in length), it has been demonstrated that hRad51 promotes homologous pairing and strand exchange reactions in vitro. Joint molecule formation is dependent upon ATP hydrolysis and DNA homology and is stimulated by the single-strand DNA-binding protein RP-A. In these reactions, the 5' terminus of the complementary strand of the linear duplex is efficiently transferred to the ssDNA. However, under standard conditions, extensive strand exchange is not observed. These results establish hRad51 as a functional homolog of RecA, but indicate that the human protein and its bacterial counterpart differ in their ability to promote extensive strand transfer. It is proposed that hRad51 mediates homology recognition and initiates strand exchange, but that extensive heteroduplex formation in higher organisms requires the actions of additional proteins (Baumann, 1996).
Human homologs of RAD50, RAD51, RAD52, RAD54 and MRE11 have been identified. Targeted disruption of the murine RAD51 gene results in an embryonic lethal phenotype, indicating that Rad51 protein is required during cell proliferation. Biochemical studies have shown that human RAD51 encodes a protein of relative molecular mass 36,966 (hRad51) that promotes ATP-dependent homologous pairing and DNA strand exchange. As a structural and functional homolog of the RecA protein from Escherichia coli, hRad51 is thought to play a central role in recombination. Yeast Rad51 has been shown to interact with Rad52 protein, as does the human homolog. hRad52 is shown to stimulate homologous pairing by hRad51. The DNA-binding properties of hRad52 indicate that Rad52 is involved in an early stage of Rad51-mediated recombination (Benson, 1998).
Yeast rad51 mutants are viable, but extremely sensitive to gamma-rays due to defective repair of double-strand breaks. In contrast, disruption of the murine RAD51 homolog is lethal, indicating an essential role of Rad51 in vertebrate cells. Clones of the chicken B lymphocyte line DT40 were generated carrying a human RAD51 transgene under the control of a repressible promoter and subsequently the endogenous RAD51 loci were disrupted. Upon inhibition of the RAD51 transgene, Rad51- cells accumulate in the G2/M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase-arrested Rad51- cells carry isochromatid-type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes (Sonada, 1998).
The repair of potentially lethal DNA double-stranded breaks (DSBs) by homologous recombination requires processing of the broken DNA into a resected DNA duplex with a protruding 3'-single-stranded DNA (ssDNA) tail. Accordingly, the canonical models for DSB repair require invasion of an intact homologous DNA template by the 3'-end of the ssDNA, a characteristic that the bacterial pairing protein RecA possesses. Unexpectedly, it has been found that for the eukaryotic homolog, Rad51 protein, the 5'-end of ssDNA is more invasive than the 3'-end. This pairing bias is unaffected by Rad52, Rad54 or Rad55-57 proteins. However, further investigation reveals that, in contrast to RecA protein, the preferred DNA substrate for Rad51 protein is not ssDNA but rather dsDNA with ssDNA tails. This important distinction permits the Rad51 proteins to promote DNA strand invasion using either 3'- or 5'-ends with similar efficiency (Mazin, 2000a).
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. 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. It is concluded 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 (Mazin, 2000b).
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. In this study, using physical criteria, the formation of Rad54-Rad51-DNA nucleoprotein co-complexes that contain equimolar amounts of each protein has been demonstrated directly. 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 (Mazin, 2003).
The bacterial RecA protein and the homologous Rad51 protein in eukaryotes both bind to single-stranded DNA (ssDNA), align it with a homologous duplex, and promote an extensive strand exchange between them. Both reactions have properties, including a tolerance of base analog substitutions that tend to eliminate major groove hydrogen bonding potential, that suggest a common molecular process underlies the DNA strand exchange promoted by RecA and Rad51. However, optimal conditions for the DNA pairing and DNA strand exchange reactions promoted by the RecA and Rad51 proteins in vitro are substantially different. When conditions are optimized independently for both proteins, RecA promotes DNA pairing reactions with short oligonucleotides at a faster rate than Rad51. For both proteins, conditions that improve DNA pairing can inhibit extensive DNA strand exchange reactions in the absence of ATP hydrolysis. Extensive strand exchange requires a spooling of duplex DNA into a recombinase-ssDNA complex, a process that can be halted by any interaction elsewhere on the same duplex that restricts free rotation of the duplex and/or complex, i.e., the reaction can get stuck. Optimization of an extensive DNA strand exchange without ATP hydrolysis requires conditions that decrease nonproductive interactions of recombinase-ssDNA complexes with the duplex DNA substrate (Rice, 2001).
Human Rad51 (hRad51), a member of a conserved family of general recombinases, is shown to have an avid capability to make DNA joints between homologous DNA molecules and promote highly efficient DNA strand exchange of the paired molecules over at least 5.4 kilobase pairs. Furthermore, maximal efficiency of homologous DNA pairing and strand exchange is strongly dependent on the heterotrimeric single-stranded DNA binding factor hRPA and requires conditions that lessen interactions of the homologous duplex with the hRad51-single-stranded DNA nucleoprotein filament. The homologous DNA pairing and strand exchange system described should be valuable for dissecting the action mechanism of hRad51 and for deciphering its functional interactions with other recombination factors (Sigurdsson, 2001).
Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is RAD51. In comparison to RecA, the bacterial homolog, human RAD51 protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. This study investigates the mechanistic basis for this stimulation. High ionic strength favors the co-aggregation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of RAD51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA-RAD51 interactions are unaffected, whereas those between RAD51 and dsDNA are destabilized. Most importantly, high salt induces a conformational change in RAD51, leading to the formation of extended nucleoprotein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA-ssDNA filament that exhibits efficient strand-exchange activity (Liu, 2004b).
Recombinase proteins assembled into helical filaments on DNA are believed to be the catalytic core of homologous recombination. The assembly, disassembly and dynamic rearrangements of this structure must drive the DNA strand exchange reactions of homologous recombination. The sensitivity of eukaryotic recombinase activity to reaction conditions in vitro suggests that the status of bound nucleotide cofactors is important for function and possibly for filament structure. Nucleoprotein filaments formed by the human recombinase Rad51 in a variety of conditions on double-stranded and single-stranded DNA were analyzed by scanning force microscopy. Regular filaments with extended double-stranded DNA correlated with active in vitro recombination, possibly due to stabilizing the DNA products of these assays. Though filaments formed readily on single-stranded DNA, they were very rarely regular structures. The irregular structure of filaments on single-stranded DNA suggests that Rad51 monomers are dynamic in filaments and that regular filaments are transient. Indeed, single molecule force spectroscopy of Rad51 filament assembly and disassembly in magnetic tweezers revealed protein association and disassociation from many points along the DNA, with kinetics different from those of RecA. The dynamic rearrangements of proteins and DNA within Rad51 nucleoprotein filaments could be key events driving strand exchange in homologous recombination (Ristic, 2005; full text of article).
NBS1 forms a complex with MRE11 and RAD50 (MRN) that is proposed to act on the upstream of two repair pathways of DNA double-strand break (DSB), homologous repair (HR) and non-homologous end joining (NHEJ). However, the function of Nbs1 in these processes has not fully been elucidated in mammals due to the lethal phenotype of cells and mice lacking Nbs1. Mouse Nbs1-null embryonic fibroblasts and embryonic stem cells were constructed through the Cre-loxP and sequential gene targeting techniques. Cells lacking Nbs1 display reduced HR of the single DSB in chromosomally integrated substrate, affecting both homology-directed repair (HDR) and single-stranded annealing pathways, and, surprisingly, increased NHEJ-mediated sequence deletion. Moreover, focus formation at DSBs and chromatin recruitment of the Nbs1 partners Rad50 and Mre11 as well as Rad51 and Brca1 are attenuated in these cells, whereas the NHEJ molecule Ku70 binding to chromatin is not affected. These data provide a novel insight into the function of MRN in the branching of DSB repair pathways (Yang, 2006).
GFP-Rad51 fusion proteins have been visualized in the nucleus of living cells to demonstrate the dynamic compartmentalization of Rad51 by self-association or by binding to BRCA2. Mutants of Rad51 that fail to oligomerize and/or to bind BRCA2 distinguish three fractions of Rad51 within the nucleoplasm: a relatively mobile fraction, an immobile oligomerized fraction, and an immobile BRCA2-bound fraction. Strikingly, inhibition of replication by hydroxyurea reduces the immobile fraction of nucleoplasmic Rad51. This effect is specific to Rad51 mutants that retain the capacity to bind BRCA2, indicating that the BRCA2-bound fraction is selectively mobilized. It is proposed that arrested replication triggers a switch between dual functions of BRCA2 in sequestering or mobilizing a small fraction of nucleoplasmic Rad51 and suggest a mechanism for the dynamic control of protein complexes that participate in homologous recombination (Yu, 2003).
BRCA2 is a breast tumor susceptibility gene encoding a 390-kDa protein with functions in maintaining genomic stability and cell cycle progression. Evidence has been accumulated to support the concept that BRCA2 has a critical role in homologous recombination of DNA double-stranded breaks by interacting with RAD51. In addition, BRCA2 may have chromatin modifying activity through interaction with a histone acetyltransferase protein, p300/CBP-associated factor (P/CAF). To explore how the functions of BRCA2 may be regulated, the post-translational modifications of BRCA2 throughout the cell cycle were examined. It was found that BRCA2 is hyperphosphorylated specifically in M phase and becomes dephosphorylated as cells exit M phase and enter interphase. This specific phosphorylation of BRCA2 was not observed in cells treated with DNA-damaging agents. Systematic mapping of the potential mitosis specific phosphorylation sites revealed the N-terminal 284 amino acids of BRCA2 (BR-N1) as the major region of phosphorylation and mass spectrometric analysis identified two phosphopeptides that contain 'phosphorylation consensus motifs' for Polo-like kinase 1 (Plk1). Phosphorylation of BR-N1 with Plk1 recapitulates the electrophoretic mobility change as seen in BR-N1 isolated from M phase cells. Plk1 interacts with BRCA2 in vivo, and mutation of Ser193, Ser205/206, and Thr203/207 to Ala in BR-N1 abolishes Plk1 phosphorylation, suggesting that BRCA2 is the substrate of Plk1. Furthermore, both the hyperphosphorylated and hypophosphorylated forms of BRCA2 bind to RAD51, whereas the M phase hyperphosphorylated form of BRCA2 no longer associates with the P/CAF, suggesting that the dissociation of P/CAF-BRCA2 complex is regulated by phosphorylation. Taken together, these results implicate a potential role of BRCA2 in modulating M phase progression (Lin, 2003).
Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. The interrelationship was studied between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, the efficiency of repair was analyzed in mammalian cells in which double-strand break (DSB) repair components were disrupted. An inverse relationship was observed between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, it was found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, it was found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. The role of RAD52 in homologous repair was examined. In contrast to yeast, Rad52-/- mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair (Stark, 2004; full text of article).
The ubiquitin (Ub)-conjugating enzyme Ubc13 is implicated in Rad6/Rad18-dependent postreplication repair (PRR) in budding yeast, but its function in vertebrates is not known. Disruption or siRNA depletion of UBC13 in chicken DT40 or human cells confers severe growth defects due to chromosome instability, and hypersensitivity to both UV and ionizing radiation, consistent with a conserved role for Ubc13 in PRR. Remarkably, Ubc13-deficient cells are also compromised for DNA double-strand break (DSB) repair by homologous recombination (HR). Recruitment and activation of the E3 Ub ligase function of BRCA1 and the subsequent formation of the Rad51 nucleoprotein filament at DSBs are abolished in Ubc13-deficient cells. Furthermore, generation of ssDNA/RPA complexes at DSBs is severely attenuated in the absence of Ubc13. These data reveal a critical and unexpected role for vertebrate Ubc13 in the initiation of HR at the level of DSB processing (Zhao, 2007).
A broad spectrum of mutations in PTEN, encoding a lipid phosphatase that inactivates the P13-K/AKT pathway, is found associated with primary tumors. Some of these mutations occur outside the phosphatase domain, suggesting that additional activities of PTEN function in tumor suppression. This study reports a nuclear function for PTEN in controlling chromosomal integrity. Disruption of Pten leads to extensive centromere breakage and chromosomal translocations. PTEN was found localized at centromeres and physically associated with CENP-C, an integral component of the kinetochore. C-terminal PTEN mutants disrupt the association of PTEN with centromeres and cause centromeric instability. Furthermore, Pten null cells exhibit spontaneous DNA double-strand breaks (DSBs). PTEN acts on chromatin and regulates expression of Rad51, which reduces the incidence of spontaneous DSBs. These results demonstrate that PTEN plays a fundamental role in the maintenance of chromosomal stability through the physical interaction with centromeres and control of DNA repair. It is proposed that PTEN acts as a guardian of genome integrity (Shen, 2007).
Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologous recombination. hRad54 is able to promote transient separation of the strands in duplex DNA via its ATP hydrolysis-driven DNA supercoiling function. The ATPase, DNA supercoiling, and DNA strand opening activities of hRad54 are greatly stimulated through an interaction with hRad51. Importantly, hRad51 and hRad54 functionally cooperate in the homologous DNA pairing reaction that forms recombination DNA intermediates. These results should provide a biochemical model for dissecting the role of hRad51 and hRad54 in recombination reactions in human cells (Sigurdsson, 2002).
In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecA-like recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, these results indicate that Rad54p is a chromatin-remodeling enzyme that promotes homologous DNA pairing events within the context of chromatin (Jaskelioff, 2003).
These results suggest that Rad54p is an extremely versatile recombination protein that plays key roles in several steps of homologous recombination. Rad54p is required for optimal recruitment of Rad51p to a double strand break in vivo, and likewise Rad54p can promote formation of the presynaptic filament in vitro by helping Rad51p contend with the inhibitory effects of the ssDNA-binding protein replication protein A.2. Several studies over the past few years have also shown that the ATPase activity of Rad54p plays key roles subsequent to formation of the presynaptic filament. For instance, Rad54p is required for the Rad51p-nucleoprotein filament to form a heteroduplex joint DNA molecule, even when the homologous donor is naked DNA. In this case, it has been proposed that Rad54p might use the free energy from ATP hydrolysis to translocate along DNA, which facilitates the homology search process. This DNA-translocation model is fully consistent with findings that Rad54p can displace a DNA triplex and that the ATPase activity of Rad54p is proportional to DNA length. Rad54p also stimulates heteroduplex DNA extension of established joint molecules. Finally, Rad54p is required for Rad51p-dependent heteroduplex joint molecule formation with a chromatin donor. In this case, the results suggest that the ATPase activity of Rad54p is used to translocate the enzyme along the nucleosomal fiber, generating superhelical torsion, which leads to enhanced nucleosomal DNA accessibility. It seems likely that this chromatin remodeling activity of Rad54p might also facilitate additional steps after heteroduplex joint formation. Future studies are now poised to reconstitute the complete homologous recombinational repair reaction that fully mimics each step in the repair of chromosomal DNA double strand breaks in vivo (Jaskelioff, 2003).
In vivo and in vitro studies have suggested the following sequence of molecular events that lead to the recombinational repair of a DSB. First, the 5' ends of DNA that flank the break are resected by an exonuclease to create ssDNA tails. Next, Rad51p polymerizes onto these DNA tails to form a nucleoprotein filament that has the capability to search for a homologous duplex DNA molecule. After DNA homology has been located, the Rad51-ssDNA nucleoprotein filament catalyzes the formation of a heteroduplex DNA joint with the homolog. The process of DNA homology search and DNA joint molecule formation is called 'homologous DNA pairing and strand exchange'. Subsequent steps entail DNA synthesis to replace the missing information followed by resolution of DNA intermediates to yield two intact duplex DNA molecules (Jaskelioff, 2003).
The homologous DNA pairing activity of Rad51p is enhanced by Rad54p. Rad54p is a member of the Swi2p/Snf2p protein family that has DNA-stimulated ATPase activity and physically interacts with Rad51p. Because of its relatedness to the Swi2p/Snf2p family of ATPases, Rad54p may have chromatin remodeling activities in addition to its established role in facilitating Rad51p-mediated homologous pairing reactions. In this study it has been shown that Rad51p and Rad54p mediate robust D-loop formation with a chromatin donor, whereas the bacterial recombinase, RecA, can only function with naked DNA. Furthermore, the ATPase activity of Rad54p is essential for D-loop formation on chromatin and Rad54p can use the free energy from ATP hydrolysis to enhance the accessibility of nucleosomal DNA. Experiments are also presented to suggest that chromatin remodeling by Rad54p and yeast SWI/SNF involves DNA translocation (Jaskelioff, 2003).
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, three distinct foci are observed that colocalize with adjacent GFP chromosome marks. The kinetics of focus formation with recombination intermediates and products were compared when HO-cleaved MATalpha 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. Evidence is presented 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 (Miyazaki, 2004; full text of article).
The efficient and accurate repair of DNA double strand breaks (DSBs) is critical to cell survival, and defects in this process can lead to genome instability and cancers. In eukaryotes, the Rad52 group of proteins dictates the repair of DSBs by the error-free process of homologous recombination (HR). A critical step in eukaryotic HR is the formation of the initial Rad51-single-stranded DNA presynaptic nucleoprotein filament. This presynaptic filament participates in a homology search process that leads to the formation of a DNA joint molecule and recombinational repair of the DSB. The Rad54 protein functions as a mediator of Rad51 binding to single-stranded DNA; this activity does not require ATP hydrolysis. A novel Rad54-dependent chromatin remodeling event has been discovered that occurs in vivo during the DNA strand invasion step of HR. This ATP-dependent remodeling activity of Rad54 appears to control subsequent steps in the HR process (Wolner, 2005; full text of article).
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. This study reports 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 (RPArfa1-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 facilitate the same reaction, demonstrating its conserved nature. The two activities of Rad52, recruiting Rad51 and annealing DNA, are coordinated in DNA strand exchange and second ssDNA capture (Sugiyama, 2006).
Werner syndrome (WS) is a rare genetic disorder characterized by genomic instability caused by defects in the WRN gene encoding a member of the human RecQ helicase family. RecQ helicases are involved in several DNA metabolic pathways including homologous recombination (HR) processes during repair of stalled replication forks. Following introduction of interstrand DNA crosslinks (ICL), WRN relocated from nucleoli to arrested replication forks in the nucleoplasm where it interacted with the HR protein RAD52. In this study, fluorescence resonance energy transfer (FRET) and immune-precipitation experiments were used to demonstrate that WRN participates in a multiprotein complex including RAD51, RAD54, RAD54B and ATR in cells where replication has been arrested by ICL. The WRN-RAD51 and WRN-RAD54B direct interaction was verified in vitro. These data support a role for WRN also in the recombination step of ICL repair (Otterlei, 2006).
Disruption of the gene encoding RAD51, the protein that catalyzes strand exchange during homologous recombination, leads to the accumulation of chromosome breaks and lethality in vertebrate cells. As RAD51 is implicated in BRCA1- and BRCA2-mediated tumor suppression as well as cellular viability, a functional analysis of a defined RAD51 mutation was performed in mammalian cells. By using a dominant negative approach, a mouse embryonic stem cell line was generated that expresses an ATP hydrolysis-defective RAD51 protein, hRAD51-K133R, at comparable levels to the endogenous wild-type RAD51 protein, whose expression is retained in these cells. It was found that these cells have increased sensitivity to the DNA-damaging agents mitomycin C and ionizing radiation and also exhibit a decreased rate of spontaneous sister-chromatid exchange. By using a reporter for the repair of a single chromosomal double-strand break, it was also found that expression of the hRAD51-K133R protein specifically inhibits homology-directed double-strand break repair. Furthermore, expression of a BRC repeat from BRCA2, a peptide inhibitor of an early step necessary for strand exchange, exacerbates the inhibition of homology-directed repair in the hRAD51-K133R expressing cell line. Thus, ATP hydrolysis by RAD51 has a key role in various types of DNA repair in mammalian cells (Stark, 2002; full text of article).
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. The current results show that Ca(2+) greatly stimulates DNA strand exchange activity of hRad51 protein. Ca(2+) exerts its stimulatory effect by modulating the ATPase activity of hRad51 protein. The data demonstrate that, in the presence of Mg(2+), 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. Ca(2+) 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; no such mechanism was found for yeast Rad51 (yRad51) protein (Bugreev, 2004).
RecA in Escherichia coli and its homolog, ScRad51 in Saccharomyces cerevisiae, are known to be essential for recombinational repair. The homolog of RecA and ScRad51 in mice, MmRad51, was mutated to determine its function. Mutant embryos arrest early during development. A decrease in cell proliferation, followed by programmed cell death and chromosome loss, was observed. Radiation sensitivity was demonstrated in trophectoderm-derived cells. Interestingly, embryonic development progressed further in a p53 null background; however, fibroblasts derived from double-mutant embryos failed to proliferate in tissue culture (Lim, 1996).
The mouse Rad51 gene is a mammalian homolog of the Escherichia coli recA and yeast RAD51 genes, both of which are involved in homologous recombination and DNA repair. To elucidate the physiological role of RAD51 protein, the gene was targeted in embryonic stem (ES) cells. Mice heterozygous for the Rad51 null mutation were intercrossed and their offspring were genotyped. There were no homozygous (Rad51-/-) pups among 148 neonates examined but a few Rad51-/- embryos were identified when examined during the early stages of embryonic development. Doubly knocked-out ES cells were not detected under conditions of selective growth. These results are interpreted to mean that RAD51 protein plays an essential role in the proliferation of cells. The homozygous Rad51 null mutation can be categorized in cell-autonomous defects. Pre-implantational lethal mutations that disrupt basic molecular functions will thus interfere with cell viability (Tsuzuki, 1996).
The nucleoprotein filament formed by Rad51 polymerization on single-stranded DNA is essential for homologous pairing and strand exchange. ATP binding is required for Rad51 nucleoprotein filament formation and strand exchange, but ATP hydrolysis is not required for these functions in vitro. Previous studies have shown that a yeast strain expressing the rad51-K191R allele is sensitive to ionizing radiation, suggesting an important role for ATP hydrolysis in vivo. The recruitment of Rad51-K191R to double-strand breaks is defective in vivo, and this phenotype can be suppressed by elimination of the Srs2 helicase, an antagonist of Rad51 filament formation. The phenotype of the rad51-K191R strain is also suppressed by overexpression of Rad54. In vitro, the Rad51-K191R protein exhibits a slight decrease in binding to DNA, consistent with the defect in presynaptic filament formation. However, the rad51-K191R mutation is dominant in heterozygous diploids, indicating that the defect is not due simply to reduced affinity for DNA. It is suggested that the Rad51-K191R protein either forms an altered filament or is defective in turnover, resulting in a reduced pool of free protein available for DNA binding (Fung, 2006).
Genomic instability is characteristic of tumor cells, and a strong correlation exists between abnormal karyotype and tumorigenicity. Increased expression of the homologous recombination and DNA repair protein Rad51 has been reported in immortalized and tumor cells, which could alter recombination pathways to contribute to the chromosomal rearrangements found in these cells. A genetic system was used to examine the potential for multiple double-strand breaks to lead to genome rearrangements in the presence of increased Rad51 expression. Analysis of repair revealed a novel class of products consistent with crossing over, involving gene conversion associated with an exchange of flanking markers leading to chromosomal translocations. Increased Rad51 also promoted aneuploidy and multiple chromosomal rearrangements. These data provide a link between elevated Rad51 protein levels, genome instability, and tumor progression (Richardson, 2004).
The phenotypically similar hamster mutants irs1 and irs1SF exhibit high spontaneous chromosome instability and broad-spectrum mutagen sensitivity, including extreme sensitivity to DNA cross-linking agents. The human XRCC2 and XRCC3 genes, which functionally complement irs1 and irs1SF, respectively, were previously mapped in somatic cell hybrids. Characterization of these genes and sequence alignments reveal that XRCC2 and XRCC3 are members of an emerging family of Rad51-related proteins that likely participate in homologous recombination to maintain chromosome stability and repair DNA damage. XRCC3 is shown to interact directly with HsRad51, and like Rad55 and Rad57 in yeast, may cooperate with HsRad51 during recombinational repair. Analysis of the XRCC2 mutation in irs1 implies that XRCC2's function is not essential for viability in cultured hamster cells (Liu, 1998).
DMC1 is a meiosis-specific gene first discovered in yeast that encodes a protein with homology to RecA and may be a component of recombination nodules. Yeast dmc1 mutants are defective in crossing over and synaptonemal complex (SC) formation, and arrest in late prophase of meiosis I. A null mutation was generated in the Dmc1 gene in mice; homozygous mutant males and females are sterile with arrest of gametogenesis in the first meiotic prophase. Chromosomes in mutant spermatocytes fail to synapse, despite the formation of axial elements that are the precursor to the SC. The strong similarity of phenotypes in Dmc1-deficient mice and yeast suggests that meiotic mechanisms have been highly conserved through evolution (Pittman, 1998).
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, homology modeling was used 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, it has been 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. It has also been 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, the binding domains of the BCDX2 and CX3 complexes have been demonstrated, the interaction of these proteins has been further demonstrated, and a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes is proposed (Miller, 2004).
During genetic recombination and the recombinational repair of chromosome breaks, DNA molecules become linked at points of strand exchange. Branch migration and resolution of these crossovers, or Holliday junctions (HJs), complete the recombination process. This study shows that extracts from cells carrying mutations in the recombination/repair genes RAD51C or XRCC3 have reduced levels of HJ resolvase activity. Moreover, depletion of RAD51C from fractionated human extracts caused a loss of branch migration and resolution activity, but these functions were restored by complementation with a variety of RAD51 paralog complexes containing RAD51C. It is concluded that the RAD51 paralogs are involved in HJ processing in human cells (Liu, 2004a).
Programmed double-strand breaks at prophase of meiosis acquire immunologically detectable RAD51-DMC1 foci or early nodules (ENs) that are associated with developing chromosome core segments; each focus is surrounded by a gammaH2AX-modified chromosome domain. The 250-300 ENs per nucleus decline in numbers during the development of full-length cores and the remaining foci are relatively evenly distributed along the mature cores. The ENs become transformed nodules (TNs) by the acquisition of RPA, BLM, MSH4 and topoisomerases that function in repair and Holliday junction resolution. At the leptotene-zygotene transition, TNs orient to positions between the aligned cores where they initiate structural interhomolog contacts prior to synaptonemal complex (SC) formation, possibly future crossover sites. Subsequently, TNs are associated with SC extension at the synaptic forks. Dephosphorylation of TN-associated histone gammaH2AX chromatin suggests annealing of single strands or repair of double-strand breaks DSBs at this time. Some 200 TNs per pachytene nucleus are distributed proportional to SC length and are evenly distributed along the SCs. At this stage, gammaH2AX-modified chromatin domains are associated with transcriptionally silenced sex chromosomes and autosomal sites. Immunogold electron microscope evidence shows that one or two TNs of the 10-15 TNs per SC acquire MLH1 protein, the hallmark of reciprocal recombination, whereas the TNs that do not acquire MLH1 protein relocate from their positions along the midline of the SCs to the periphery of the SCs. Relocation of TNs may be associated with the conversion of potential crossovers into non-crossovers (Moens, 2007).
RAD51C is a member of the RecA/RAD51 protein family, which is known to play an important role in DNA repair by homologous recombination. In mice, it is essential for viability. Therefore, a hypomorphic allele of Rad51c was generated in addition to a null allele. A subset of mice expressing the hypomorphic allele is infertile. This infertility is caused by sexually dimorphic defects in meiotic recombination, revealing its two distinct functions. Spermatocytes undergo a developmental arrest during the early stages of meiotic prophase I, providing evidence for the role of RAD51C in early stages of RAD51-mediated recombination. In contrast, oocytes can progress normally to metaphase I after superovulation but display precocious separation of sister chromatids, aneuploidy, and broken chromosomes at metaphase II. These defects suggest a possible late role of RAD51C in meiotic recombination. Based on the marked reduction in Holliday junction (HJ) resolution activity in Rad51c-null mouse embryonic fibroblasts, it is proposed that this late function may be associated with HJ resolution (Kuznetsov, 2007).
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; 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. This study shows that, like MLH1, RAD51C localizes to mouse meiotic chromosomes at pachytene/diplotene. Using immunoprecipitation and gel filtration analyses, it was found that Holliday junction resolvase activity associates 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 (Liu, 2007).
Members of the RecQ helicase family play critical roles in genome maintenance. There are five RecQ homologs in mammals, and defects in three of these (BLM, WRN, and RECQL4) give rise to cancer predisposition syndromes in humans. RECQL and RECQL5 have not been associated with a human disease. This study shows that deletion of Recql5 in mice results in cancer susceptibility. Recql5-deficient cells exhibit elevated frequencies of spontaneous DNA double-strand breaks and homologous recombination (HR) as scored using a reporter that harbors a direct repeat, and are prone to gross chromosomal rearrangements in response to replication stress. To understand how RECQL5 regulates HR, purified proteins were used to demonstrate that human RECQL5 binds the Rad51 recombinase and inhibits Rad51-mediated D-loop formation. By biochemical means and electron microscopy, it was shown that RECQL5 displaces Rad51 from single-stranded DNA (ssDNA) in a reaction that requires ATP hydrolysis and RPA. Together, these results identify RECQL5 as an important tumor suppressor that may act by preventing inappropriate HR events via Rad51 presynaptic filament disruption (Hu, 2007).
Homologous recombination (HR) is a fundamental molecular process in all organisms. In meiosis, HR is necessary for the proper segregation of homologous chromosomes and generates genetic diversity through the shuffling of parental alleles. In mitotic cells, HR is an important pathway for repairing chromosomal breaks and gaps, and for restarting damaged or stalled DNA replication forks. However, inappropriate or untimely HR events can have mutagenic and oncogenic consequences. For example, reciprocal exchanges (crossovers) between homologous chromosomes can lead to somatic loss of heterozygosity (LOH), while crossovers between nonhomologous chromosomes can result in translocation. Also, crossovers between repeated sequences on the same chromosome can result in deletions or inversions. For these reasons, specific mechanisms have evolved for regulating HR to minimize these potentially deleterious rearrangements (Hu, 2007).
Genetic analyses in Saccharomyces cerevisiae indicate that two DNA helicases, Srs2 and Sgs1, function in different pathways to suppress crossover events in mitotic cells. Mutations in either Srs2 or Sgs1 result in a hyperrecombination phenotype. In humans, mutations in BLM, which encodes the human ortholog of Sgs1, give rise to the rare hereditary disorder Bloom's syndrome. This disorder is marked by an elevated rate of sister chromatid exchange (SCE), increased chromosomal instability, and a high incidence of cancer. BLM suppresses SCEs by acting in conjunction with the Type 1A topoisomerase, Topo IIIα, and a recently identified protein, BLAP75, to mediate the dissolution of double Holliday junctions (DHJ; a late HR intermediate), a process that yields solely noncrossover recombinants. Like its human counterpart, Sgs1 forms a complex with the Top3 topoisomerase and Rmi1 (the BLAP75 ortholog), suggesting that it might function to suppress SCEs by a similar mechanism (Hu, 2007).
Srs2 is a superfamily 1 helicase with similarities to the bacterial UvrD/Rep helicases. The mechanistic basis of the Srs2 function was elucidated by biochemical studies that revealed its ability to bind Rad51 and to dismantle the Rad51-ssDNA (single-stranded DNA) nucleoprotein filament, the key catalytic intermediate in recombination reactions. To date, no apparent Srs2 ortholog has been identified in other eukaryotes, although the recently identified Fbh1 helicase shows some structural similarity to the Srs2 helicase family and studies in Schizosaccharomyces pombe suggest that Fbh1 plays a role in processing HR intermediates. However, Fbh1-deficient DT40 cells show no prominent sensitivity to DNA damaging agents, and exhibit only a mild SCE phenotype. This raises the question as to whether attenuation of HR by disruption of the Rad51 presynaptic filament represents a significant mechanism for HR regulation in higher eukaryotes (Hu, 2007).
Sgs1 and BLM are members of the RecQ family of DNA helicases. Sgs1 is the sole RecQ helicase in budding yeast. Interestingly, humans have a total of five RecQ helicase encoding genes (RECQL, BLM, WRN, RECQL4, and RECQL5). They share a conserved seven-motif helicase domain but are otherwise distinct from one another by their unique amino acid composition outside the helicase domain, suggesting that they have related but different roles. In addition to BLM, defects in both WRN and RECQL4 are also associated with heritable genome instability and cancer disorders. Therefore, while BLM likely represents the Sgs1 ortholog, the other RECQ-like helicases represent potential candidates as the functional equivalent of Srs2 in humans. Mouse cells deficient in the RECQL5 homolog Recql5 exhibit an elevated level of SCEs, thus implicating this helicase in the regulation of HR. Importantly, deletion of both Recql5 and Blm further increases the SCE frequency, consistent with Recql5 acting to regulate SCEs in mitotic cells via a mechanism that is distinct from Blm, perhaps by functioning similarly to Srs2 to suppress the channeling of DNA lesions into HR (Hu, 2007).
This paper shows that deletion of Recql5 in mice results in increased susceptibility to cancer. Recql5-deficient cells exhibit elevated frequencies of spontaneous double-strand breaks (DSBs) and HR between direct repeats, and are prone to the accumulation of gross chromosomal rearrangements (GCRs) in response to replication stress. Moreover, by biochemical means, a mechanistic basis by which RECQL5 functions in suppressing GCRs and tumorigenesis is provided. Specifically, human RECQL5 binds the Rad51 recombinase, and a catalytic quantity of this helicase inhibits Rad51-mediated D-loop formation markedly. Furthermore, RECQL5 displaces Rad51 from ssDNA in a reaction that requires ATP hydrolysis by RECQL5 and is stimulated by the ssDNA-binding protein RPA. Taken together, these data provide compelling evidence that this unique member of the RecQ helicase family functions to minimize the propensity of oncogenic rearrangements by suppressing the accumulation of DSBs and attenuating HR by disrupting the Rad51 presynaptic filament (Hu, 2007).
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