RAD51-associated protein 1 (RAD51AP1) interacts with the meiotic recombinase DMC1 through a conserved motif

Homologous recombination (HR) reactions mediated by the RAD51 recombinase are essential for DNA and replication fork repair, genome stability, and tumor suppression. RAD51-associated protein 1 (RAD51AP1) is an important HR factor that associates with and stimulates the recombinase activity of RAD51. We have recently shown that RAD51AP1 also partners with the meiotic recombinase DMC1, displaying isoform-specific interactions with DMC1. Here, we have characterized the DMC1 interaction site in RAD51AP1 by a series of truncations and point mutations to uncover a highly conserved WVPP motif critical for DMC1 interaction but dispensable for RAD51 association. This RAD51AP1 motif is reminiscent of the FVPP motif in the tumor suppressor protein BRCA2 that mediates DMC1 interaction. These results further implicate RAD51AP1 in meiotic HR via RAD51 and DMC1.     

Role of human RAD51 recombinase in the cycle checkpoint and survival of a cell

The RAD 51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a significant role in both mitotic and meiotic homologous recombination. Here, we demonstrate that short-term silencing of the Rad51 gene by specific small interfering RNA (siRNA) that inhibits cell proliferation and reduces the viability of most cells. Cells with suppressed expression of Rad51 gene have altered cell cycles and accumulate in the S and G₂ phases. Our findings show that the disruption of homologous recombination leads to cell death. However, some cells, e.g., MCF-7 cells, are insensitive to the suppression of Rad51 gene expression.     

Promotion of homologous recombination and genomic stability by RAD51AP1 via RAD51 recombinase enhancement

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.     

Two modules in the BRC repeats of BRCA2 mediate structural and functional interactions with the RAD51 recombinase

The breast and ovarian cancer suppressor protein BRCA2 controls the RAD51 recombinase in reactions that lead to homologous DNA recombination (HDR). BRCA2 binds RAD51 via eight conserved BRC repeat motifs of approximately 35 amino acids, each with a varying capacity to bind RAD51. BRC repeats both promote and inhibit RAD51 assembly on different DNA substrates to regulate HDR, but the structural basis for these functions is unclear. Here, we demarcate two tetrameric clusters of hydrophobic residues in the BRC repeats, interacting with distinct pockets in RAD51, and show that the co-location of both modules within a single BRC repeat is necessary for BRC–RAD51 binding and function. The two modules comprise the sequence FxxA, known to inhibit RAD51 assembly by blocking the oligomerization interface, and a previously unrecognized tetramer with the consensus sequence LFDE, which binds to a RAD51 pocket distinct from this interface. The LFDE motif is essential in BRC repeats for modes of RAD51 binding both permissive and inhibitory to RAD51 oligomerization. Targeted insertion of point mutations in RAD51 that disrupt the LFDE-binding pocket impair its assembly at DNA damage sites in living cells. Our findings suggest a model for the modular architecture of BRC repeats that provides fresh insight into the mechanisms regulating homologous DNA recombination.     

Subunit interface residues F129 and H294 of human RAD51 are essential for recombinase function

RAD51 mediated homologous recombinational repair (HRR) of DNA double-strand breaks (DSBs) is essential to maintain genomic integrity. RAD51 forms a nucleoprotein filament (NPF) that catalyzes the fundamental homologous pairing and strand exchange reaction (recombinase) required for HRR. Based on structural and functional homology with archaeal and yeast RAD51, we have identified the human RAD51 (HsRAD51) subunit interface residues HsRad51(F129) in the Walker A box and HsRad51(H294) in the L2 ssDNA binding region as potentially important participants in salt-induced conformational transitions essential for recombinase activity. We demonstrate that the HsRad51(F129V) and HsRad51(H294V) substitution mutations reduce DNA dependent ATPase activity and are largely defective in the formation of a functional NPF, which ultimately eliminates recombinase catalytic functions. Our data are consistent with the conclusion that the HsRAD51(F129) and HsRAD51(H294) residues are important participants in the cation-induced allosteric activation of HsRAD51.     

Roles of XRCC2, RAD51B and RAD51D in RAD51-Independent SSA Recombination

The repair of DNA double-strand breaks by recombination is key to the maintenance of genome integrity in all living organisms. Recombination can however generate mutations and chromosomal rearrangements, making the regulation and the choice of specific pathways of great importance. In addition to end-joining through non-homologous recombination pathways, DNA breaks are repaired by two homology-dependent pathways that can be distinguished by their dependence or not on strand invasion catalysed by the RAD51 recombinase. Working with the plant Arabidopsis thaliana, we present here an unexpected role in recombination for the Arabidopsis RAD51 paralogues XRCC2, RAD51B and RAD51D in the RAD51-independent single-strand annealing pathway. The roles of these proteins are seen in spontaneous and in DSB-induced recombination at a tandem direct repeat recombination tester locus, both of which are unaffected by the absence of RAD51. Individual roles of these proteins are suggested by the strikingly different severities of the phenotypes of the individual mutants, with the xrcc2 mutant being the most affected, and this is confirmed by epistasis analyses using multiple knockouts. Notwithstanding their clearly established importance for RAD51-dependent homologous recombination, XRCC2, RAD51B and RAD51D thus also participate in Single-Strand Annealing recombination.     

RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51.

RAD51B and RAD51C are two of five known paralogs of the human RAD51 protein that are thought to function in both homologous recombination and DNA double-strand break repair. This work describes the in vitro and in vivo identification of the RAD51B/RAD51C heterocomplex. The RAD51B/RAD51C heterocomplex was isolated and purified by immunoaffinity chromatography from insect cells co-expressing the recombinant proteins. Moreover, co-immunoprecipitation of the RAD51B and RAD51C proteins from HeLa, MCF10A, and MCF7 cells strongly suggests the existence of an endogenous RAD51B/RAD51C heterocomplex. We extended these observations to examine the interaction between the RAD51B/RAD51C complex and the other RAD51 paralogs. Immunoprecipitation using protein-specific antibodies showed that RAD51C is central to a single large protein complex and/or several smaller complexes with RAD51B, RAD51D, XRCC2, and XRCC3. However, our experiments showed no evidence for the inclusion of RAD51 within these complexes. Further analysis is required to elucidate the function of the RAD51B/RAD51C heterocomplex and its association with the other RAD51 paralogs in the processes of homologous recombination and DNA double-strand break repair.     

Homologous recombination properties of OsRad51, a recombinase from rice

cDNA corresponding to OsRad51 protein was isolated from cDNA library of rice flowers (Oryza sativa, Indica cultivar group) and cloned in to pET28a expression vector. The protein was over expressed in E. coli BL21 (DE3) and purified. Purified OsRad51 could bind single and double stranded DNA, however it showed higher affinity for single stranded DNA. Transmission Electron Microscopy (TEM) studies of OsRad51-DNA complexes showed that this protein formed ring like structures and bound DNA forming filaments. OsRad51 protein promoted renaturation of complementary single strands in to duplex DNA molecules and also showed ATPase activity, which was stimulated by single strand DNA. Fluorescence resonance energy transfer (FRET) assays revealed that OsRad51 promoted homology dependent renaturation as well as strand exchange reactions. Renaturation activity was ATP dependent; however strand exchange activity was ATP independent. This is the first report on in vitro characterization of Rad51 protein from crop plants.     

Endogenous mutations in human uncoupling protein 3 alter its functional properties

Human uncoupling protein (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative phosphorylation and is a candidate gene for obesity. Expression of native human UCP3 mutations in yeast showed complete loss (R70W), significant reduction (R143X), or no effect (V102I and IVS6+1G > A) on the uncoupling activity of UCP3. It is concluded that certain mutations in UCP3 alter its functional impact on membrane potential (deltaphi), possibly conferring susceptibility to develop metabolic diseases.     

Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51

Bloom's syndrome (BS) is an autosomal recessive disorder that predisposes individuals to a wide range of cancers. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. The precise role played by these enzymes in the cell remains to be determined. However, genome-wide hyper-recombination is a feature of many RecQ helicase-deficient cells. In eukaryotes, a central step in homologous recombination is catalyzed by the RAD51 protein. In response to agents that induce DNA double-strand breaks, RAD51 accumulates in nuclear foci that are thought to correspond to sites of recombinational repair. Here, we report that purified BLM and human RAD51 interact in vitro and in vivo, and that residues in the N- and C-terminal domains of BLM can independently mediate this interaction. Consistent with these observations, BLM localizes to a subset of RAD51 nuclear foci in normal human cells. Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to ionizing radiation. Nevertheless, the formation of RAD51 foci does not require functional BLM. Indeed, in untreated BS cells, an abnormally high proportion of the cells contain RAD51 nuclear foci. Exogenous expression of BLM markedly reduces the fraction of cells containing RAD51 foci. The interaction between BLM and RAD51 appears to have been evolutionarily conserved since the C-terminal domain of Sgs1, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Rad51. Furthermore, genetic analysis reveals that the SGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway. Potential roles for BLM in the RAD51 recombinational repair pathway are discussed.     

RAD54 family translocases counter genotoxic effects of RAD51 in human tumor cells

The RAD54 family DNA translocases have several biochemical activities. One activity, demonstrated previously for the budding yeast translocases, is ATPase-dependent disruption of RAD51-dsDNA binding. This activity is thought to promote dissociation of RAD51 from heteroduplex DNA following strand exchange during homologous recombination. In addition, previous experiments in budding yeast have shown that the same activity of Rad54 removes Rad51 from undamaged sites on chromosomes; mutants lacking Rad54 accumulate nonrepair-associated complexes that can block growth and lead to chromosome loss. Here, we show that human RAD54 also promotes the dissociation of RAD51 from dsDNA and not ssDNA. We also show that translocase depletion in tumor cell lines leads to the accumulation of RAD51 on chromosomes, forming complexes that are not associated with markers of DNA damage. We further show that combined depletion of RAD54L and RAD54B and/or artificial induction of RAD51 overexpression blocks replication and promotes chromosome segregation defects. These results support a model in which RAD54L and RAD54B counteract genome-destabilizing effects of direct binding of RAD51 to dsDNA in human tumor cells. Thus, in addition to having genome-stabilizing DNA repair activity, human RAD51 has genome-destabilizing activity when expressed at high levels, as is the case in many human tumors.     

Overexpression of human RAD51 and RAD52 reduces double-strand break-induced homologous recombination in mammalian cells

Double-strand breaks (DSBs) can be repaired by homologous recombination (HR) in mammalian cells, often resulting in gene conversion. RAD51 functions with RAD52 and other proteins to effect strand exchange during HR, forming heteroduplex DNA (hDNA) that is resolved by mismatch repair to yield a gene conversion tract. In mammalian cells RAD51 and RAD52 overexpression increase the frequency of spontaneous HR, and one study indicated that overexpression of mouse RAD51 enhances DSB-induced HR in Chinese hamster ovary (CHO) cells. We tested the effects of transient and stable overexpression of human RAD51 and/or human RAD52 on DSB-induced HR in CHO cells and in human cells. DSBs were targeted to chromosomal recombination substrates with I-SceI nuclease. In all cases, excess RAD51 and/or RAD52 reduced DSB-induced HR, contrasting with prior studies. These distinct results may reflect differences in recombination substrate structures or different levels of overexpression. Excess RAD51/RAD52 did not increase conversion tract lengths, nor were product spectra otherwise altered, indicating that excess HR proteins can have dominant negative effects on HR initiation, but do not affect later steps such as hDNA formation, mismatch repair or the resolution of intermediates.     

Distribution of the Rad51 recombinase in human and mouse spermatocytes.

In vitro, the human Rad51 protein (hRad51) promotes homologous pairing and strand exchange reactions suggestive of a key role in genetic recombination. To analyse its role in this process, polyclonal antibodies raised against hRad51 were used to study the distribution of Rad51 in human and mouse spermatocytes during meiosis I. In human spermatocytes, hRad51 was found to form discrete nuclear foci from early zygotene to late pachytene. The foci always co-localized with lateral element proteins, components of the synaptonemal complex (SC). During zygotene, the largest foci were present in regions undergoing synapsis, suggesting that Rad51 is a component of early recombination nodules. Pachytene nuclei showed a greatly reduced level of Rad51 labelling, with the exceptions of any asynapsed autosomes and XY segments, which were intensely labelled. The distribution of Rad51 in mouse spermatocytes was similar to that found in human spermatocytes, except that in this case Rad51 was detectable at leptotene. From these results, we conclude that the Rad51 protein has a role in the interhomologue interactions that occur during meiotic recombination. These interactions are spatially and temporally associated with synapsis during meiotic prophase I.     

Cooperation of RAD51 and RAD54 in regression of a model replication fork

DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the 'template-strand switch' mechanism. This mechanism involves replication fork regression into a Holliday junction ('chicken foot structure'), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion.     

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.     

Novel selenoproteins identified in silico and in vivo by using a conserved RNA structural motif

Selenocysteine is incorporated into selenoproteins by an in-frame UGA codon whose readthrough requires the selenocysteine insertion sequence (SECIS), a conserved hairpin in the 3'-untranslated region of eukaryotic selenoprotein mRNAs. To identify new selenoproteins, we developed a strategy that obviates the need for prior amino acid sequence information. A computational screen was used to scan nucleotide sequence data bases for sequences presenting a potential SECIS secondary structure. The computer-selected hairpins were then assayed in vivo for their functional capacities, and the cDNAs corresponding to the SECIS winners were identified. Four of them encoded novel selenoproteins as confirmed by in vivo experiments. Among these, SelZf1 and SelZf2 share a common domain with mitochondrial thioredoxin reductase-2. The three proteins, however, possess distinct N-terminal domains. We found that another protein, SelX, displays sequence similarity to a protein involved in bacterial pilus formation. For the first time, four novel selenoproteins were discovered based on a computational screen for the RNA hairpin directing selenocysteine incorporation.     

Structural and torsional properties of the RAD51-dsDNA nucleoprotein filament

Human RAD51 is a key protein in the repair of DNA by homologous recombination. Its assembly onto DNA, which induces changes in DNA structure, results in the formation of a nucleoprotein filament that forms the basis of strand exchange. Here, we determine the structural and mechanical properties of RAD51-dsDNA filaments. Our measurements use two recently developed magnetic tweezers assays, freely orbiting magnetic tweezers and magnetic torque tweezers, designed to measure the twist and torque of individual molecules. By directly monitoring changes in DNA twist on RAD51 binding, we determine the unwinding angle per RAD51 monomer to be 45°, in quantitative agreement with that of its bacterial homolog, RecA. Measurements of the torque that is built up when RAD51-dsDNA filaments are twisted show that under conditions that suppress ATP hydrolysis the torsional persistence length of the RAD51-dsDNA filament exceeds that of its RecA counterpart by a factor of three. Examination of the filament’s torsional stiffness for different combinations of divalent ions and nucleotide cofactors reveals that the Ca²⁺ ion, apart from suppressing ATPase activity, plays a key role in increasing the torsional stiffness of the filament. These quantitative measurements of RAD51-imposed DNA distortions and accumulated mechanical stress suggest a finely tuned interplay between chemical and mechanical interactions within the RAD51 nucleoprotein filament.     

Sequence, chromosomal location and expression analysis of the murine homologue of human RAD51L2/RAD51C.

The Rad51 protein has been shown to play a vital role in the DNA repair process. In humans, its interaction with proteins like BRCA1 and BRCA2 has provided an insight into the mechanism of how these molecules function as tumor suppressors. Several members of the Rad51-like family have been recently identified, including RAD51L2. This gene has been found to be amplified in breast tumors suggesting its role in tumor progression. Here, we describe the cloning of the murine homologue of the human RAD51L2/RAD51C gene. Sequence analysis has revealed that the murine Rad51l2 protein is 86% identical and 93% similar to its human homologue. In spite of such high sequence conservation, the murine protein lacks the first nine amino acids present in the human protein. We have cloned and confirmed the sequence of the 5' end of the murine Rad51l2 cDNA using 5' RACE technique as well as by sequencing the genomic region flanking the first exon of the murine Rad51l2 gene. Northern analysis shows that Rad51l2 is expressed in several adult tissues as well as in embryos at various developmental stages. The murine Rad51l2 gene maps to chromosome 11 and is located in the syntenic region of human chromosome 17q22-23, where the human RAD51L2 is present.