FBH1 Helicase Disrupts RAD51 Filaments in Vitro and Modulates Homologous Recombination in Mammalian Cells

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.     

Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.

The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. We show that 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, we have found that Rad51 protein is 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.     

Defects in recombination activity caused by somatic and germline mutations in the multimerization/BRCA2 binding region of human RAD51 protein

The human RAD51 recombinase possesses DNA pairing and strand exchange activities that are essential for the error-free, homology-directed repair of DNA double-strand breaks. The recombination activities of RAD51 are activated upon its assembly into presynaptic filaments on single-stranded DNA at resected DSB ends. Defects in filament assembly caused by mutations in RAD51 or its regulators such as BRCA2 are associated with human cancer. Here we describe two novel RAD51 missense variants located in the multimerization/BRCA2 binding region of RAD51. F86L is a breast tumor-derived somatic variant that affects the interface between adjacent RAD51 protomers in the presynaptic filament. E258A is a germline variant that maps to the interface region between the N-terminal and RecA homology domains of RAD51. Both variants exhibit abnormal biochemistry including altered DNA strand exchange activity. Both variants inhibit the DNA strand exchange activity of wild-type RAD51, suggesting a mechanism for negative dominance. The inhibitory effect of F86L on wild-type RAD51 is surprising since F86L alone exhibits robust DNA strand exchange activity. Our findings indicate that even DNA strand exchange-proficient variants can have negative functional interactions with wild-type RAD51. Thus heterozygous F86L or E258 mutations in RAD51 could promote genomic instability, and thereby contribute to tumor progression.     

Biochemical characterization of the human RAD51 protein. I. ATP hydrolysis

The prototypical bacterial RecA protein promotes recombination/repair by catalyzing strand exchange between homologous DNAs. While the mechanism of strand exchange remains enigmatic, ATP-induced cooperativity between RecA protomers is critical for its function. A human RecA homolog, human RAD51 protein (hRAD51), facilitates eukaryotic recombination/repair, although its ability to hydrolyze ATP and/or promote strand exchange appears distinct from the bacterial RecA. We have quantitatively examined the hRAD51 ATPase. The catalytic efficiency (k(cat)/K(m)) of the hRAD51 ATPase was approximately 50-fold lower than the RecA ATPase. Altering the ratio of DNA/hRAD51 and including salts that stimulate DNA strand exchange (ammonium sulfate and spermidine) were found to affect the catalytic efficiency of hRAD51. The average site size of hRAD51 was determined to be approximately 3 nt (bp) for both single-stranded and double-stranded DNA. Importantly, hRAD51 lacks the magnitude of ATP-induced cooperativity that is a hallmark of RecA. Together, these results suggest that hRAD51 may be unable to coordinate ATP hydrolysis between neighboring protomers.     

Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD51 recombinase

Human RAD51 protein catalyzes DNA pairing and strand exchange reactions that are central to homologous recombination and homology-directed DNA repair. Successful recombination/repair requires the formation of a presynaptic filament of RAD51 on ssDNA. Mutations in BRCA2 and other proteins that control RAD51 activity are associated with human cancer. Here we describe a set of mutations associated with human breast tumors that occur in a common structural motif of RAD51. Tumor-associated D149N, R150Q and G151D mutations map to a Schellman loop motif located on the surface of the RecA homology domain of RAD51. All three variants are proficient in DNA strand exchange, but G151D is slightly more sensitive to salt than wild-type (WT). Both G151D and R150Q exhibit markedly lower catalytic efficiency for adenosine triphosphate hydrolysis compared to WT. All three mutations alter the physical properties of RAD51 nucleoprotein filaments, with G151D showing the most dramatic changes. G151D forms mixed nucleoprotein filaments with WT RAD51 that have intermediate properties compared to unmixed filaments. These findings raise the possibility that mutations in RAD51 itself may contribute to genome instability in tumor cells, either directly through changes in recombinase properties, or indirectly through changes in interactions with regulatory proteins.     

Recombinational repair in yeast: functional interactions between Rad51 and Rad54 proteins.

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. Here we demonstrate by genetic, molecular and biochemical criteria that Rad51 and Rad54 proteins 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 was 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. Our data provide compelling evidence that Rad51 and Rad54 proteins interact in vivo and that this interaction is functionally important for recombinational DNA damage repair. As both proteins are conserved throughout evolution from yeasts to humans, a similar protein-protein interaction may be expected in other organisms.     

Regulation of DNA strand exchange in homologous recombination

Homologous recombination, the exchange of DNA strands between homologous DNA molecules, is involved in repair of many structural diverse DNA lesions. This versatility stems from multiple ways in which homologous DNA strands can be rearranged. At the core of homologous recombination are recombinase proteins such as RecA and RAD51 that mediate homology recognition and DNA strand exchange through formation of a dynamic nucleoprotein filament. Four stages in the life cycle of nucleoprotein filaments are filament nucleation, filament growth, homologous DNA pairing and strand exchange, and filament dissociation. Progression through this cycle requires a sequence of recombinase-DNA and recombinase protein-protein interactions coupled to ATP binding and hydrolysis. The function of recombinases is controlled by accessory proteins that allow coordination of strand exchange with other steps of homologous recombination and that tailor to the needs of specific aberrant DNA structures undergoing recombination. Accessory proteins are also able to reverse filament formation thereby guarding against inappropriate DNA rearrangements. The dynamic instability of the recombinase-DNA interactions allows both positive and negative action of accessory proteins thereby ensuring that genome maintenance by homologous recombination is not only flexible and versatile, but also accurate.     

ATP hydrolysis by mammalian RAD51 has a key role during homology-directed DNA repair

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, we have begun a functional analysis of a defined RAD51 mutation in mammalian cells. By using a dominant negative approach, we generated a mouse embryonic stem cell line 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. We 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, we 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.     

The Essential Functions of Human Rad51 Are Independent of ATP Hydrolysis

Genetic recombination and the repair of double-strand DNA breaks in Saccharomyces cerevisiae require Rad51, a homologue of the Escherichia coli RecA protein. In vitro, Rad51 binds DNA to form an extended nucleoprotein filament and catalyzes the ATP-dependent exchange of DNA between molecules with homologous sequences. Vertebrate Rad51 is essential for cell proliferation. Using site-directed mutagenesis of highly conserved residues of human Rad51 (hRad51) and gene targeting of the RAD51 locus in chicken DT40 cells, we examined the importance of Rad51's highly conserved ATP-binding domain. Mutant hRad51 incapable of ATP hydrolysis (hRad51K-133R) binds DNA less efficiently than the wild type but catalyzes strand exchange between homologous DNAs. hRad51 does not need to hydrolyze ATP to allow vertebrate cell proliferation, form nuclear foci, or repair radiation-induced DNA damage. However, cells expressing hRad51K-133R show greatly reduced targeted integration frequencies. These findings show that ATP hydrolysis is involved in DNA binding by hRad51 and suggest that the extent of DNA complexed with hRad51 in nucleoprotein influences the efficiency of recombination.     

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.     

Structural analysis of an RNase T1 variant with an altered guanine binding segment

Information concerning the function of recombination proteins in mammalian cells has been obtained from biochemical studies, but little is known about their mechanisms of action in growing cells. The eukaryotic recombination protein RAD51, a homologue of the Escherichia coli RecA protein, has been shown to interact with various proteins, including the p53 protein, the guardian of genomic stability maintenance. Here, the hamster RAD51 protein, CgRAD51, has been overexpressed in the SPD8 cell line, derived from Chinese hamster V79 cells. This cell line offers unique possibilities for studying different mechanisms for homologous recombination on endogenous substrates. We report that the SPD8 cell line contains a mutated p53 gene, which provides new insights into the recombination process in these cells. The present study demonstrates that overexpression of CgRAD51 in these cells results in a two- to threefold increase in endogenous recombination. In addition, sequence analysis indicated that RAD51 promotes homologous recombination by a chromatid exchange mechanism.     

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.     

RAD51 variant proteins from human lung and kidney tumors exhibit DNA strand exchange defects

In human cells, error-free repair of DNA double-strand breaks requires the DNA pairing and strand exchange activities of RAD51 recombinase. Activation of RAD51 recombination activities requires the assembly of RAD51 presynaptic filaments on the single-stranded DNA that forms at resected DSB ends. Mutations in proteins that control presynaptic filament assembly, such as BRCA2, and in RAD51 itself, are associated with human breast cancer. Here we describe the properties of two mutations in RAD51 protein that derive from human lung and kidney tumors, respectively. Sequence variants Q268P and Q272L both map to the DNA binding loop 2 (L2) region of RAD51, a motif that is involved in DNA binding and in the allosteric activation of ATP hydrolysis and DNA strand exchange activities. Both mutations alter the thermal stability, DNA binding, and ATPase properties of RAD51, however both variants retain intrinsic DNA strand exchange activity towards oligonucleotide substrates under optimized conditions. In contrast, both Q268P and Q272L variants exhibit drastically reduced DNA strand exchange activity in reaction mixtures containing long homologous ssDNA and dsDNA substrates and human RPA protein. Mixtures of wild-type and variant proteins also exhibit reduced DNA strand exchange activity, suggesting that heterozygous mutations could negatively affect DNA recombination and repair processes in vivo. Together, the findings of this study suggest that hypomorphic missense mutations in RAD51 protein could be drivers of genomic instability in cancer cells, and thereby contribute to the etiology of metastatic disease.     

A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity

Accurate DNA double-strand break repair through homologous recombination is essential for preserving genome integrity. Disruption of the gene encoding RAD51, the protein that catalyzes DNA strand exchange during homologous recombination, results in lethality of mammalian cells. Proteins required for homologous recombination, also play an important role during DNA replication. To explore the role of RAD51 in DNA replication and DSB repair, we used a knock-in strategy to express a carboxy-terminal fusion of green fluorescent protein to mouse RAD51 (mRAD51-GFP) in mouse embryonic stem cells. Compared to wild-type cells, heterozygous mRad51^+/wt-GFP embryonic stem cells showed increased sensitivity to DNA damage induced by ionizing radiation and mitomycin C. Moreover, gene targeting was found to be severely impaired in mRad51^+/wt-GFP embryonic stem cells. Furthermore, we found that mRAD51-GFP foci were not stably associated with chromatin. From these experiments we conclude that this mRad51-GFP allele is an antimorphic allele. When this allele is present in a heterozygous condition over wild-type mRad51, embryonic stem cells are proficient in DNA replication but display defects in homologous recombination and DNA damage repair.     

Overexpression of RAD51 suppresses recombination defects: a possible mechanism to reverse genomic instability

RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA-binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the ‘recombination mediators’. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as ‘recombination co-mediators’. Defects in either recombination mediators or co-mediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic re-stabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51 expression.     

Arabidopsis RAD51C gene is important for homologous recombination in meiosis and mitosis

Rad51 is a homolog of the bacterial RecA recombinase, and a key factor in homologous recombination in eukaryotes. Rad51 paralogs have been identified from yeast to vertebrates. Rad51 paralogs are thought to play an important role in the assembly or stabilization of Rad51 that promotes homologous pairing and strand exchange reactions. We previously characterized two RAD51 paralogous genes in Arabidopsis (Arabidopsis thaliana) named AtRAD51C and AtXRCC3, which are homologs of human RAD51C and XRCC3, respectively, and described the interaction of their products in a yeast two-hybrid system. Recent studies showed the involvement of AtXrcc3 in DNA repair and functional role in meiosis. To determine the role of RAD51C in meiotic and mitotic recombination in higher plants, we characterized a T-DNA insertion mutant of AtRAD51C. Although the atrad51C mutant grew normally during vegetative developmental stage, the mutant produced aborted siliques, and their anthers did not contain mature pollen grains. Crossing of the mutant with wild-type plants showed defective male and female gametogeneses as evidenced by lack of seed production. Furthermore, meiosis was severely disturbed in the mutant. The atrad51C mutant also showed increased sensitivity to gamma-irradiation and cisplatin, which are known to induce double-strand DNA breaks. The efficiency of homologous recombination in somatic cells in the mutant was markedly reduced relative to that in wild-type plants.     

RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates

DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes. Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining. A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates. Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence. The dependence of recombinational gap repair on genes belonging to the RAD52 epistasis group was tested in this system. RAD51, RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors. No homologous recombination products were recovered from rad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, or RAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not on RAD51. The residual repair events in rad51 mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms for RAD51-dependent and RAD51-independent events are different. Plasmid gap repair was reduced synergistically in rad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair.     

DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression.

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.     

The Caenorhabditis elegansRAD51 homolog is transcribed into two alternative mRNAs potentially encoding proteins of different sizes

In prokaryotes, the RecA protein plays a pivotal role in homologous recombination, catalyzing the transfer of a single DNA strand into an homologous molecule. Structural homologs of the bacterial RecA protein, called Rad51, have been found in different eukaryotes (from yeast to man), suggesting a certain level of conservation in recombination pathways among living organisms. We have cloned the homolog of RAD51 in Caenorhabditis elegans. The CeRAD51 gene is transcribed into two alternative mRNAs and potentially codes for two proteins of 395 and 357 amino acids in length, respectively. We discuss the evolutionary implications of these findings.