Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair

In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the Rad51-dependent recombination pathway. These paralogs form two complexes (Rad51C/Xrcc3 and Rad51B/C/D/Xrcc2), which play roles in somatic recombination, DNA repair and chromosome stability. However, little is known of their possible involvement in meiosis, due to the inviability of the corresponding knockout mice. We have recently reported that the Arabidopsis homolog of one of these Rad51 paralogs (AtXrcc3) is involved in DNA repair and meiotic recombination and present here Arabidopsis lines carrying mutations in three other Rad51 paralogs (AtRad51B, AtRad51C and AtXrcc2). Disruption of any one of these paralogs confers hypersensitivity to the DNA cross-linking agent Mitomycin C, but not to gamma-irradiation. Moreover, the atrad51c-1 mutant is the only one of these to show meiotic defects similar to those of the atxrcc3 mutant, and thus only the Rad51C/Xrcc3 complex is required to achieve meiosis. These results support conservation of functions of the Rad51 paralogs between vertebrates and plants and differing requirements for the Rad51 paralogs in meiosis and DNA repair.     

Analysis of the relationships between ATM and the Rad54 paralogs involved in homologous recombination repair

Ataxia-telangiectasia is a pleiotropic genomic instability disorder caused by lack or inactivation of the ATM protein kinase and characterized by progressive ataxia, immunodeficiency, ionizing radiation sensitivity and cancer predisposition. ATM mobilizes the cellular response to DNA double strand breaks by phosphorylating key players in this response. Double strand breaks are repaired by either nonhomologous end-joining or homologous recombination (HR) in which the Rad54 and Rad54B paralogs function. Here, we investigated the functional relationships between Atm and the Rad54 proteins by constructing compound genotypes in mice. Mouse strains were generated that combined inactivation of the Atm, Rad54 and Rad54B genes. All mutant genotypes were viable, but obtained at sub-Mendelian ratios. Double mutants for Atm and each Rad54 paralog exhibited reduced body weight and shorter lifespan, but no distinct neurological phenotype. Concomitant inactivation of ATM and Rad54 did not increase IR sensitivity; however, the triple Atm/Rad54/Rad54B mutant exhibited a significant IR hypersensitivity compared to the other genotypes. Interestingly, Atm?/? animals also exhibited hypersensitivity to the crosslinking agent mitomycin C, which was increased by deficiency of either one of the Rad54 paralogs. Our results reveal a differential interaction of the ATM-mediated DNA damage response and Rad54 paralog-mediated HR depending on the DNA damaging agent that initiates the response.     

Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52

Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.     

Homologous DNA pairing by human recombination factors Rad51 and Rad54

Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologous recombination. We show an ability of hRad54 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, we demonstrate that hRad51 and hRad54 functionally cooperate in the homologous DNA pairing reaction that forms recombination DNA intermediates. Our results should provide a biochemical model for dissecting the role of hRad51 and hRad54 in recombination reactions in human cells.     

Coordinated response of mammalian Rad51 and Rad52 to DNA damage

Biochemical analysis has shown that mammalian Rad51 and Rad52 interact and synergize in DNA recombination reactions in vitro, but these proteins have not been shown to function together in response to DNA damage in vivo. By analysis of murine cells expressing murine Rad52 tagged with green fluorescent protein (GFP)–Rad52, we now show that DNA damage causes Rad51 and GFP–Rad52 to colocalize in distinct nuclear foci. Cells expressing GFP–Rad52 show both increased survival and an increased number of Rad51 foci, raising the possibility that Rad52 is limiting for repair. These observations provide evidence of coordinated function of Rad51 and Rad52 in vivo and support the hypothesis that Rad52 plays an important role in the DNA damage response in mammalian cells.     

Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54

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.     

Novel and diverse functions of the DNA mismatch repair family in mammalian meiosis and recombination

The mismatch repair (MMR) family is a highly conserved group of proteins that function in genome stabilization and mutation avoidance. Their role has been particularly well studied in the context of DNA repair following replication errors, and disruption of these processes results in characteristic microsatellite instability, repair defects and, in mammals, susceptibility to cancer. An additional role in meiotic recombination has been described for several family members, as revealed by extensive studies in yeast. More recently, the role of the mammalian MMR family in meiotic progression has been elucidated by the phenotypic analysis of mice harboring targeted mutations in the genes encoding several MMR family members. This review will discuss the phenotypes of the various mutant mouse lines and, drawing from our knowledge of MMR function in yeast meiosis and in somatic cell repair, will attempt to elucidate the significance of MMR activity in mouse germ cells. These studies highlight the importance of comparative analysis of MMR orthologs across species, and also underscore distinct sexually dimorphic characteristics of mammalian recombination and meiosis.     

DNA repair,recombination, and damage signaling

DNA must be accurately copied and propagated from one cell division to the next, and from one generation to the next. To ensure the faithful transmission of the genome, a plethora of distinct as well as overlapping DNA repair and recombination pathways have evolved. These pathways repair a large variety of lesions, including alterations to single nucleotides and DNA single and double-strand breaks, that are generated as a consequence of normal cellular function or by external DNA damaging agents. In addition to the proteins that mediate DNA repair, checkpoint pathways have also evolved to monitor the genome and coordinate the action of various repair pathways. Checkpoints facilitate repair by mediating a transient cell cycle arrest, or through initiation of cell suicide if DNA damage has overwhelmed repair capacity. In this chapter, we describe the attributes of Caenorhabditis elegans that facilitate analyses of DNA repair, recombination, and checkpoint signaling in the context of a whole animal. We review the current knowledge of C. elegans DNA repair, recombination, and DNA damage response pathways, and their role during development, growth, and in the germ line. We also discuss how the analysis of mutational signatures in C. elegans is helping to inform cancer mutational signatures in humans.     

Rad54, the motor of homologous recombination

Homologous recombination (HR) performs crucial functions including DNA repair, segregation of homologous chromosomes, propagation of genetic diversity, and maintenance of telomeres. HR is responsible for the repair of DNA double-strand breaks and DNA interstrand cross-links. The process of HR is initiated at the site of DNA breaks and gaps and involves a search for homologous sequences promoted by Rad51 and auxiliary proteins followed by the subsequent invasion of broken DNA ends into the homologous duplex DNA that then serves as a template for repair. The invasion produces a cross-stranded structure, known as the Holliday junction. Here, we describe the properties of Rad54, an important and versatile HR protein that is evolutionarily conserved in eukaryotes. Rad54 is a motor protein that translocates along dsDNA and performs several important functions in HR. The current review focuses on the recently identified Rad54 activities which contribute to the late phase of HR, especially the branch migration of Holliday junctions.     

The mitotic DNA damage checkpoint proteins Rad17 and Rad24 are required for repair of double-strand breaks during meiosis in yeast

We show here that deletion of the DNA damage checkpoint genes RAD17 and RAD24 in Saccharomyces cerevisiae delays repair of meiotic double-strand breaks (DSBs) and results in an altered ratio of crossover-to-noncrossover products. These mutations also decrease the colocalization of immunostaining foci of the RecA homologs Rad51 and Dmc1 and cause a delay in the disappearance of Rad51 foci, but not of Dmc1. These observations imply that RAD17 and RAD24 promote efficient repair of meiotic DSBs by facilitating proper assembly of the meiotic recombination complex containing Rad51. Consistent with this proposal, extra copies of RAD51 and RAD54 substantially suppress not only the spore inviability of the rad24 mutant, but also the gamma-ray sensitivity of the mutant. Unexpectedly, the entry into meiosis I (metaphase I) is delayed in the checkpoint single mutants compared to wild type. The control of the cell cycle in response to meiotic DSBs is also discussed.     

In vivo roles of Rad52, Rad54, and Rad55 proteins in Rad51-mediated recombination

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. Here we used chromatin immunoprecipitation to monitor the in vivo association of Saccharomyces cerevisiae Rad51p with both the cleaved MATa locus and the HML alpha 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 HML alpha. Lack of Rad54p does not significantly impair Rad51p recruitment to MAT or its initial association with HML alpha; however, Rad54p is required at or before the initiation of DNA synthesis after synapsis has occurred at the 3' end of the invading strand.     

Interaction of human recombination proteins Rad51 and Rad54.

The cDNA for human protein HsRad54, which is a structural homolog of Saccharomyces cerevisiae recombination/repair protein Rad54, was cloned and expressed in Escherichia coli. As demonstrated by analysis in vitro and in vivo, HsRad54 protein interacts with human Rad51 recombinase. The interaction is mediated by the N-terminal domain of HsRad54 protein, which interacts with both free and DNA-bound HsRad51 protein.     

Homologous recombination is involved in repair of chromium-induced DNA damage in mammalian cells

Chromium is a potent human carcinogen, probably because of its well-documented genotoxic effects. Chromate (Cr[VI]) causes a wide range of DNA lesions, including DNA crosslinks and strand breaks, presumably due to the direct and indirect effects of DNA oxidation. Homologous recombination repair (HRR) is important for error-free repair of lesions occurring at replication forks. Here, we show that HR deficient cell lines irs1SF and V-C8, deficient in XRCC3 and BRCA2, respectively, are hypersensitive to Cr[VI], implicating this repair pathway in repair of Cr[VI] damage. Furthermore, we find that Cr[VI] causes DNA double-strand breaks and triggers both Rad51 foci formation and induction of HRR. Collectively, these data suggest that HRR is important in repair of Cr[VI]-induced DNA damage. In addition, we find that ERCC1, XRCC1 and DNA-PKcs defective cells are hypersensitive to Cr[VI], indicating that several repair pathways cooperate in repairing Cr[VI]-induced DNA damage.     

The response of mammalian cells to UV-light reveals Rad54-dependent and independent pathways of homologous recombination

Ultraviolet (UV) radiation-induced DNA lesions can be efficiently repaired by nucleotide excision repair (NER). However, NER is less effective during replication of UV-damaged chromosomes. In contrast, translesion DNA synthesis (TLS) and homologous recombination (HR) are capable of dealing with lesions in replicating DNA. The core HR protein in mammalian cells is the strand exchange protein RAD51, which is aided by numerous proteins, including RAD54. We used RAD54 as a cellular marker for HR to study the response of mammalian embryonic stem (ES) cells to UV irradiation. In contrast to yeast, ES cells lacking RAD54 are not UV sensitive. Here we show that the requirement for mammalian RAD54 is masked by active NER. By genetically inactivating NER and HR through disruption of the Xpa and Rad54 genes, respectively, we demonstrate the contribution of HR to chromosomal integrity upon UV irradiation. We demonstrate using chromosome fiber analysis at the individual replication fork level, that HR activity is important for the restart of DNA replication after induction of DNA damage by UV-light in NER-deficient cells. Furthermore, our data reveal RAD54-dependent and -independent contributions of HR to the cellular sensitivity to UV-light, and they uncover that RAD54 can compensate for the loss of TLS polymerase η with regard to UV-light sensitivity. In conclusion, we show that HR is important for the progression of UV-stalled replication forks in ES cells, and that protection of the fork is an interplay between HR and TLS.     

DNA homologous recombination repair in mammalian cells

DNA double-strand breaks (DSBs) are the most serious DNA damage. Due to a great variety of factors causing DSBs, the efficacy of their repair is crucial for the cell's functioning and prevents DNA fragmentation, chromosomal translocation and deletion. In mammalian cells DSBs can be repaired by non-homologous end joining (NHEJ), homologous recombination (HRR) and single strand annealing (SSA). HRR can be divided into the first and second phase. The first phase is initiated by sensor proteins belonging to the MRN complex, that activate the ATM protein which target HRR proteins to obtain the second response phase--repair. HRR is precise because it utilizes a non-damaged homologous DNA fragment as a template. The key players of HRR in mammalian cells are MRN, RPA, Rad51 and its paralogs, Rad52 and Rad54.     

Homologous recombination in DNA repair and DNA damage tolerance

Homologous recombination （HR） comprises a series of interrelated pathways that function in the repair of DNA double-stranded breaks （DSBs） and interstrand crosslinks （ICLs）. In addition, recombination provides critical support for DNA replication in the recovery of stalled or broken replication forks, contributing to tolerance of DNA damage. A central core of proteins, most critically the RecA homolog Rad51, catalyzes the key reactions that typify HR： homology search and DNA strand invasion. The diverse functions of recombination are reflected in the need for context-specific factors that perform supplemental functions in conjunction with the core proteins. The inability to properly repair complex DNA damage and resolve DNA replication stress leads to genomic instability and contributes to cancer etiology. Mutations in the BRCA2 recombination gene cause predisposition to breast and ovarian cancer as well as Fanconi anemia, a cancer predisposition syndrome characterized by a defect in the repair of DNA interstrand crosslinks. The cellular functions of recombination are also germane to DNA-based treatment modalities of cancer, which target replicating cells by the direct or indirect induction of DNA lesions that are substrates for recombination pathways. This review focuses on mechanistic aspects of HR relating to DSB and ICL repair as well as replication fork support.     

RAD51 paralogs: roles in DNA damage signalling, recombinational repair and tumorigenesis

Chromosomal double-strand breaks (DSBs) have the potential to permanently arrest cell cycle progression and endanger cell survival. They must therefore be efficiently repaired to preserve genome integrity and functionality. Homologous recombination (HR) provides an important error-free mechanism for DSB repair in mammalian cells. In addition to RAD51, the central recombinase activity in mammalian cells, a family of proteins known as the RAD51 paralogs and consisting of five proteins (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3), play an essential role in the DNA repair reactions through HR. The RAD51 paralogs act to transduce the DNA damage signal to effector kinases and to promote break repair. However, their precise cellular functions are not fully elucidated. Here we discuss recent advances in our understanding of how these factors mediate checkpoint responses and act in the HR repair process. In addition, we highlight potential functional similarities with the BRCA2 tumour suppressor, through the recently reported links between RAD51 paralog deficiencies and tumorigenesis triggered by genome instability.     

Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination

During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.