XRCC3 is required for efficient repair of chromosome breaks by homologous recombination

XRCC3 was originally identified as a human gene able to complement the DNA damage sensitivity, chromosomal instability and impaired growth of the mutant hamster cell line irs1SF. More recently, it has been cloned, sequenced and found to bear sequence homology to the highly conserved eukaryotic repair and recombination gene RAD51. The phenotype of irs1SF and the identification of XRCC3 as a member of the RAD51 gene family have suggested a role for XRCC3 in repair of DNA damage by homologous recombination. Homologous recombinational repair (HRR) of a specifically induced chromosomal double-strand break (DSB) was assayed in irs1SF cells with and without transient complementation by human XRCC3. Complementation with XRCC3 increased the frequencies of repair by 34- to 260-fold. The results confirm a role for XRCC3 in HRR of DNA DSB, and the importance of this repair pathway for the maintenance of chromosomal integrity in mammalian cells.     

Role of mammalian RAD51L2 (RAD51C) in recombination and genetic stability

The highly conserved RAD51 protein has a central role in homologous recombination. Five novel RAD51-like genes have been identified in mammalian cells, but little is known about their functions. A DNA damage-sensitive hamster cell line, irs3, was found to have a mutation in the RAD51L2 gene and an undetectable level of RAD51L2 protein. Resistance of irs3 to DNA-damaging agents was significantly increased by expression of the human RAD51L2 gene, but not by other RAD51-like genes or RAD51 itself. Consistent with a role for RAD51L2 in homologous recombination, irs3 cells show a reduction in sister chromatid exchange, an increase in isochromatid breaks, and a decrease in damage-dependent RAD51 focus formation compared with wild type cells. As recently demonstrated for human cells, we show that RAD51L2 forms part of two separate complexes of hamster RAD51-like proteins. Strikingly, neither complex of RAD51-like proteins is formed in irs3 cells. Our results demonstrate that RAD51L2 has a key role in mammalian RAD51-dependent processes, contingent on the formation of protein complexes involved in homologous recombination repair.     

The importance of XRCC2 in RAD51-related DNA damage repair

The repair of DNA damage by homologous recombination (HR) is a key pathway for the maintenance of genetic stability in mammalian cells, especially during and following DNA replication. The central HR protein is RAD51, which ensures high fidelity DNA repair by facilitating strand exchange between damaged and undamaged homologous DNA segments. Several RAD51-like proteins, including XRCC2, appear to help with this process, but their roles are not well understood. Here we show that XRCC2 is highly conserved and that most substantial truncations of the protein destroy its ability to function. XRCC2 and its partner protein RAD51L3 are found to interact with RAD51 in the 2-hybrid system, and XRCC2 is shown to be important but not essential for the accumulation of RAD51 at the sites of DNA damage. We visualize the localization of XRCC2 protein at the same sites of DNA damage for the first time using specialized irradiation conditions. Our data indicate that an important function of XRCC2 is to enhance the activity of RAD51, so that the loss of XRCC2 results in a severe delay in the early response of RAD51 to DNA damage.     

RAD51B plays an essential role during somatic and meiotic recombination in Physcomitrella

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are the strand-exchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essential to the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.     

Differential roles of XRCC2 in homologous recombinational repair of stalled replication forks

Homologous recombination is an important mechanism in DNA replication to ensure faithful DNA synthesis and genomic stability. In this study, we investigated the role of XRCC2, a member of the RAD51 paralog family, in cellular recovery from replication arrest via homologous recombination. The protein expression of XRCC2, as well as its binding partner RAD51D, is dramatically increased in S- and G2-phases, suggesting that these proteins function during and after DNA synthesis. XRCC2 mutant irs1 cells exhibit hypersensitivity to hydroxyurea (HU) and are defective in the induction of RAD51 foci after HU treatment. In addition, the HU-induced chromatin association of RAD51 is deficient in irs1 mutant. Interestingly, irs1 cells are only slightly sensitive to thymidine and able to form intact RAD51 foci in S-phase cells arrested with thymidine. Irs1 cells showed increased level of chromatin-bound RAD51 as well as the wild type cells after thymidine treatment. Both HU and thymidine induce gamma-H2AX foci in arrested S-phase nuclei. These results suggest that XRCC2 is involved in repair of HU-induced damage, but not thymidine-induced damage, at the stalled replication forks. Our data suggest that there are at least two sub-pathways in homologous recombination, XRCC2-dependent and -independent, for repair of stalled replication forks and assembly of RAD51 foci following replication arrest in S-phase.     

The splicing-factor related protein SFPQ/PSF interacts with RAD51D and is necessary for homology-directed repair and sister chromatid cohesion

DNA double-stranded breaks (DSBs) are among the most severe forms of DNA damage and responsible for chromosomal translocations that may lead to gene fusions. The RAD51 family plays an integral role in preserving genome stability by homology directed repair of DSBs. From a proteomics screen, we recently identified SFPQ/PSF as an interacting partner with the RAD51 paralogs, RAD51D, RAD51C and XRCC2. Initially discovered as a potential RNA splicing factor, SFPQ was later shown to have homologous recombination and non-homologous end joining related activities and also to bind and modulate the function of RAD51. Here, we demonstrate that SFPQ interacts directly with RAD51D and that deficiency of both proteins confers a severe loss of cell viability, indicating a synthetic lethal relationship. Surprisingly, deficiency of SFPQ alone also leads to sister chromatid cohesion defects and chromosome instability. In addition, SFPQ was demonstrated to mediate homology directed DNA repair and DNA damage response resulting from DNA crosslinking agents, alkylating agents and camptothecin. Taken together, these data indicate that SFPQ association with the RAD51 protein complex is essential for homologous recombination repair of DNA damage and maintaining genome integrity.     

The RAD51 family member, RAD51L3, is a DNA-stimulated ATPase that forms a complex with XRCC2

The Rad51 protein in eukaryotic cells is a structural and functional homolog of Escherichia coli RecA with a role in DNA repair and genetic recombination. Several proteins showing sequence similarity to Rad51 have previously been identified in both yeast and human cells. In Saccharomyces cerevisiae, two of these proteins, Rad55p and Rad57p, form a heterodimer that can stimulate Rad51-mediated DNA strand exchange. Here, we report the purification of one of the representatives of the RAD51 family in human cells. We demonstrate that the purified RAD51L3 protein possesses single-stranded DNA binding activity and DNA-stimulated ATPase activity, consistent with the presence of "Walker box" motifs in the deduced RAD51L3 sequence. We have identified a protein complex in human cells containing RAD51L3 and a second RAD51 family member, XRCC2. By using purified proteins, we demonstrate that the interaction between RAD51L3 and XRCC2 is direct. Given the requirements for XRCC2 in genetic recombination and protection against DNA-damaging agents, we suggest that the complex of RAD51L3 and XRCC2 is likely to be important for these functions in human cells.     

The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair

The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.     

A transient assay for recombination demonstrates that Arabidopsis SNM1 and XRCC3 enhance non-homologous recombination

Replacement of endogenous genes by homologous recombination is rare in plants; the majority of genetic modifications are the result of transforming DNA molecules undergoing random genomic insertion by way of non-homologous recombination. Factors that affect chromatin remodeling and DNA repair are thought to have the potential to enhance the frequency of homologous recombination in plants. Conventional tools to study the frequencies of genetic recombination often rely on stable transformation-based approaches, with these systems being rarely capable of high-throughput or combinatorial analysis. We developed a series of vectors that use chemiluminescent (LUC and REN) reporter genes to assay the relative frequency of homologous and non-homologous recombination in plants. These transient assay vectors were used to screen 14 candidate genes for their effects on recombination frequencies in Nicotiana benthamiana plants. Over-expression of Arabidopsis genes with sequence similarity to SNM1 from yeast and XRCC3 from humans enhanced the frequency of non-homologous recombination when assayed using two different donor vectors. Transient N. benthamiana leaf systems were also used in an alternative assay for preliminary measurements of homologous recombination frequencies, which were found to be enhanced by over-expression of RAD52, MIM and RAD51 from yeast, as well as CHR24 from Arabidopsis. The findings for the assays described here are in line with previous studies that analyzed recombination frequencies using stable transformation. The assays we report have revealed functions in non-homologous recombination for the Arabidopsis SNM1 and XRCC3 genes, so the suppression of these genes' expression offers a potential means to enhance the gene targeting frequency in plants. Furthermore, our findings also indicate that plant gene targeting frequencies could be enhanced by over-expression of RAD52, MIM, CHR24, and RAD51 genes.     

Tetratricopeptide-motif-mediated interaction of FANCG with recombination proteins XRCC3 and BRCA2

Fanconi anaemia is an inherited chromosomal instability disorder characterised by cellular sensitivity to DNA interstrand crosslinkers, bone-marrow failure and a high risk of cancer. Eleven FA genes have been identified, one of which, FANCD1, is the breast cancer susceptibility gene BRCA2. At least eight FA proteins form a nuclear core complex required for monoubiquitination of FANCD2. The BRCA2/FANCD1 protein is connected to the FA pathway by interactions with the FANCG and FANCD2 proteins, both of which co-localise with the RAD51 recombinase, which is regulated by BRCA2. These connections raise the question of whether any of the FANC proteins of the core complex might also participate in other complexes involved in homologous recombination repair. We therefore tested known FA proteins for direct interaction with RAD51 and its paralogs XRCC2 and XRCC3. FANCG was found to interact with XRCC3, and this interaction was disrupted by the FA-G patient derived mutation L71P. FANCG was co-immunoprecipitated with both XRCC3 and BRCA2 from extracts of human and hamster cells. The FANCG-XRCC3 and FANCG-BRCA2 interactions did not require the presence of other FA proteins from the core complex, suggesting that FANCG also participates in a DNA repair complex that is downstream and independent of FANCD2 monoubiquitination. Additionally, XRCC3 and BRCA2 proteins co-precipitate in both human and hamster cells and this interaction requires FANCG. The FANCG protein contains multiple tetratricopeptide repeat motifs (TPRs), which function as scaffolds to mediate protein-protein interactions. Mutation of one or more of these motifs disrupted all of the known interactions of FANCG. We propose that FANCG, in addition to stabilising the FA core complex, may have a role in building multiprotein complexes that facilitate homologous recombination repair.     

RAD51C germline mutations in breast and ovarian cancer cases from high-risk families

BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5' UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001). Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene.     

Identification of functional domains in the RAD51L2 (RAD51C) protein and its requirement for gene conversion

The RAD51 protein plays a key part in the process of homologous recombination through its catalysis of homologous DNA pairing and strand exchange. Additionally five novel mammalian RAD51-like proteins have been identified in mammalian cells, but their roles in homologous recombination are much less well established. These RAD51-like proteins form two different complexes, but only the RAD51L2 (RAD51C) protein is a part of both complexes. By using site-directed mutagenesis of RAD51L2, we show that non-conservative mutation of the putative ATP-binding domain severely reduces its function, whereas a conservative mutation shows partial loss of function. We find that the protein is localized to the nucleus by tagging RAD51L2 with the green fluorescent protein and provisionally identify a C-terminal domain that acts as a nuclear localization signal. Further, a RAD51L2-deficient cell line was found to have significantly reduced homology-directed repair of a DNA double-strand break by gene conversion. This recombination defect could be partially restored by ectopic expression of the human RAD51L2 protein. Therefore we have identified protein domains that are important for the correct functioning of RAD51L2 and have shown that there is a specific requirement for RAD51L2 in gene conversion in mammalian cells.     

c-ABL tyrosine kinase stabilizes RAD51 chromatin association

The assembly of RAD51 recombinase on DNA substrates at sites of breakage is essential for their repair by homologous recombination repair (HRR). The signaling pathway that triggers RAD51 assembly at damage sites to form subnuclear foci is unclear. Here, we provide evidence that c-ABL, a tyrosine kinase activated by DNA damage which phosphorylates RAD51 on Tyr-315, works at a previously unrecognized, proximal step to initiate RAD51 assembly. We first show that c-ABL associates with chromatin after DNA damage in a manner dependent on its kinase activity. Using RAD51 mutants that are unable to oligomerize to form a nucleoprotein filament, we separate RAD51 assembly on DNA to form foci into two steps: stable chromatin association followed by oligomerization. We show that phosphorylation on Tyr-315 by c-ABL is required for chromatin association of oligomerization-defective RAD51 mutants, but is insufficient to restore oligomerization. Our findings suggest a new model for the regulation of early steps of HRR.     

Effects of XRCC2 and RAD51B mutations on somatic and meiotic recombination in Arabidopsis thaliana

Homologous recombination is key to the maintenance of genome integrity and the creation of genetic diversity. At the mechanistic level, recombination involves the invasion of a homologous DNA template by broken DNA ends, repair of the break and exchange of genetic information between the two DNA molecules. Invasion of the template in eukaryotic cells is catalysed by the RAD51 and DMC1 recombinases, assisted by a number of accessory proteins, including the RAD51 paralogues. Eukaryotic genomes encode a variable number of RAD51 paralogues, ranging from two in yeast to five in animals and plants. The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilization of the RAD51‐DNA nucleofilament. Somatic recombination assays and immunocytology confirm that the three ‘non‐meiotic’ paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and that they are not required for the formation of radioinduced RAD51 foci. Given the presence of all five proteins in meiotic cells, the apparent absence of a meiotic role for RAD51B, RAD51D and XRCC2 is surprising, and perhaps simply the result of a more subtle meiotic phenotype in the mutants. Analysis of meiotic recombination confirms this, showing that the absence of XRCC2, and to a lesser extent RAD51B, but not RAD51D, increases rates of meiotic crossing over. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells.     

Cells expressing murine RAD52 splice variants favor sister chromatid repair

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

RNF138 interacts with RAD51D and is required for DNA interstrand crosslink repair and maintaining chromosome integrity

The RAD51 family is integral for homologous recombination (HR) mediated DNA repair and maintaining chromosome integrity. RAD51D, the fourth member of the family, is a known ovarian cancer susceptibility gene and required for the repair of interstrand crosslink DNA damage and preserving chromosomal stability. In this report, we describe the RNF138 E3 ubiquitin ligase that interacts with and ubiquitinates the RAD51D HR protein. RNF138 is a member of an E3 ligase family that contains an amino-terminal RING finger domain and a putative carboxyl-terminal ubiquitin interaction motif. In mammalian cells, depletion of RNF138 increased the stability of the RAD51D protein, suggesting that RNF138 governs ubiquitin-proteasome-mediated degradation of RAD51D. However, RNF138 depletion conferred sensitivity to DNA damaging agents, reduced RAD51 focus formation, and increased chromosomal instability. Site-specific mutagenesis of the RNF138 RING finger domain demonstrated that it was necessary for RAD51D ubiquitination. Presence of RNF138 also enhanced the interaction between RAD51D and a known interacting RAD51 family member XRCC2 in a yeast three-hybrid assay. Therefore, RNF138 is a newly identified regulatory component of the HR mediated DNA repair pathway that has implications toward understanding how ubiquitination modifies the functions of the RAD51 paralog protein complex.     

Effect of mutations in genes affecting homologous recombination on restriction enzyme-mediated and illegitimate recombination in Saccharomyces cerevisiae.

Restriction enzyme-mediated events (REM events; integration of transforming DNA catalyzed by in vivo action of a restriction enzyme) and illegitimate recombination events (IR events; integration of transforming DNA that shares no homology with the host genomic sequences) have been previously characterized in Saccharomyces cerevisiae. This study determines the effect of mutations in genes that are involved in homologous recombination and/or in the repair of double-stranded DNA breaks on these recombination events. Surprisingly, REM events are completely independent of the double-strand-break repair functions encoded by the RAD51, RAD52, and RAD57 genes but require the RAD50 gene product. IR events are under different genetic control than homologous integration events. In the rad50 mutant, homologous integration occurred at wild-type frequency, whereas the frequency of IR events was 20- to 100-fold reduced. Conversely, the rad52 mutant was grossly deficient in homologous integration (at least 1,000-fold reduced) but showed only a 2- to 8-fold reduction in IR frequency.     

The Rad51 paralog Rad51B promotes homologous recombinational repair

The highly conserved Saccharomyces cerevisiae Rad51 protein plays a central role in both mitotic and meiotic homologous DNA recombination. Seven members of the Rad51 family have been identified in vertebrate cells, including Rad51, Dmc1, and five Rad51-related proteins referred to as Rad51 paralogs, which share 20 to 30% sequence identity with Rad51. In chicken B lymphocyte DT40 cells, we generated a mutant with RAD51B/RAD51L1, a member of the Rad51 family, knocked out. RAD51B(-/-) cells are viable, although spontaneous chromosomal aberrations kill about 20% of the cells in each cell cycle. Rad51B deficiency impairs homologous recombinational repair (HRR), as measured by targeted integration, sister chromatid exchange, and intragenic recombination at the immunoglobulin locus. RAD51B(-/-) cells are quite sensitive to the cross-linking agents cisplatin and mitomycin C and mildly sensitive to gamma-rays. The formation of damage-induced Rad51 nuclear foci is much reduced in RAD51B(-/-) cells, suggesting that Rad51B promotes the assembly of Rad51 nucleoprotein filaments during HRR. These findings show that Rad51B is important for repairing various types of DNA lesions and maintaining chromosome integrity.     

Interplay between human DNA repair proteins at a unique double-strand break in vivo

DNA repair by homologous recombination is essential for preserving genomic integrity. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) play important roles in this process. In this study, we show that human RAD51 interacts with RAD51C-XRCC3 or RAD51B-C-D-XRCC2. In addition to being critical for RAD51 focus formation, RAD51C localizes to DNA damage sites. Inhibition of RAD51C results in a decrease in cellular proliferation consistent with a role in repairing double-strand breaks (DSBs) that occur naturally. To monitor a single DNA repair event, we developed immunofluorescence and chromatin immunoprecipitation (ChIP) methods on human cells where a unique DSB can be created in vivo. Using this system, we observed a single focus of RAD51C, RAD51 and 53BP1, which colocalized with gamma-H2AX. ChIPs revealed that endogenous human RAD51, RAD51C, RAD51D, XRCC2, XRCC3 and MRE11 proteins are recruited in the S-G2 phase of the cell cycle, while Ku80 is recruited during G1. We propose that RAD51C ensures a tight regulation of RAD51 assembly during DSB repair and plays a direct role in repairing DSBs in vivo.