Mutagenesis Advance Access originally published online on October 19, 2005
Mutagenesis 2005 20(6):433-440; doi:10.1093/mutage/gei059
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The ATPase motif in RAD51D is required for resistance to DNA interstrand crosslinking agents and interaction with RAD51C
Department of Physiology and Cardiovascular Genomics, Medical University of Ohio, Toledo, OH, USA and 1Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA, USA
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Homologous recombination (HR) is a mechanism for repairing DNA interstrand crosslinks and double-strand breaks. In mammals, HR requires the activities of the RAD51 family (RAD51, RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1), each of which contains conserved ATP binding sequences (Walker Motifs A and B). RAD51D is a DNA-stimulated ATPase that interacts directly with RAD51C and XRCC2. To test the hypothesis that ATP binding and hydrolysis by RAD51D are required for the repair of interstrand crosslinks, site-directed mutations in Walker Motif A were generated, and complementation studies were performed in Rad51d-deficient mouse embryonic fibroblasts. The K113R and K113A mutants demonstrated a respective 96 and 83% decrease in repair capacity relative to wild-type. Further examination of these mutants, by yeast two-hybrid analyses, revealed an 8-fold reduction in the ability to associate with RAD51C whereas interaction with XRCC2 was retained at a level similar to the S111T control. These cell-based studies are the first evidence that ATP binding and hydrolysis by RAD51D are required for efficient HR repair of DNA interstrand crosslinks.
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Introduction |
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Homologous recombination (HR) is responsible for suppressing the formation of chromosome abnormalities and repairing DNA double-strand breaks and interstrand crosslinks [reviewed in (1
–3)]. This high fidelity repair process uses homologous sequence to restore damaged genetic information and suppress extensive loss of heterozygosity (4). Mammalian proteins involved in HR repair include BRCA1, BRCA2, RAD51 (a RecA homolog) and the RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). The RAD51 paralogs interact to form at least two stable complexes (a dimer consisting of RAD51C–XRCC3 and a larger complex containing RAD51B, RAD51C, RAD51D and XRCC2) that assist the RAD51 strand transferase during HR (5–8). With the exception of Dmc1, which is expressed during gamete formation, disruption of each of the Rad51 genes results in increased sensitivity to DNA damage and a genome instability phenotype characterized by various chromosomal abnormalities including gaps, breaks and translocations (9–13). Correlations between altered expression of HR genes in various malignancies and the increased prevalence of allelic variants in cancer patients suggest that defects in this repair mechanism play a role in carcinogenesis (14–17).
Each of the RAD51 paralogs contains two highly conserved ATP binding motifs: Motif A (GXXXXGK(T)XXXXXXI/V) and Motif B, (R/KXXXGXXXL) followed by a series of hydrophobic residues, where X is any amino acid (18). Conservative substitution of the lysine in Walker Motif A is predicted to result in a protein capable of limited binding, but not hydrolysis, of ATP (19). The loss of a positively charged residue at this position will probably abolish ATP binding (20,21). Accordingly, a lysine to arginine substitution in the Walker Motif A of RecA confers cellular sensitivity to DNA damage and a reduction in genetic recombination without affecting strand exchange activity (22,23). Expression of the analogous mutant of human RAD51 (K133R) results in a dominant inhibitory effect in murine embryonic stem cells characterized by increased sensitivity to DNA damaging agents, decreased spontaneous sister-chromatid exchange and reduced HR repair (24). Mutation of the Walker Motifs in RAD51C and XRCC3 results in decreased ability to repair the DNA interstrand crosslinks introduced by mitomycin C (MMC) (25–27).
Not all proteins mediating the RAD51 strand transferase require an ability to bind and hydrolyze ATP. Walker Motif A mutants of yeast Rad55 (lysine 49 substituted with arginine or alanine) display increased X-ray sensitivity and sporulation defects at 23°C, but the identical substitutions in the consensus lysine of Rad57 have no physiological effect (28). Furthermore, though XRCC2 is required for the formation of normal RAD51 foci, mutation of Motif A does not affect its role in DNA repair (29). RAD51B binds DNA and displays ATPase activity, although mutation of its Walker motifs has not been reported (30).
Understanding the effect of ATP hydrolysis on the assembly of DNA repair complexes is critical in elucidation of the double-strand break repair process (31). Both RecA and RAD51 undergo conformational changes during ATP binding or hydrolysis that regulate their polymerization on single-stranded DNA (32–35). Evidence is now emerging to suggest that RAD51-related protein complexes are also controlled by this small molecule effector. ATP has been shown to stimulate the binding of RAD51D–XRCC2 to single-stranded DNA (36), and ATP hydrolysis is necessary for normal dynamics of the RAD51C–XRCC3 complex (26).
RAD51D is unique among the RAD51 paralogs owing to the diversity of its physiological roles. In addition to its association with other RAD51 family members, it is the only paralog currently known to support telomere maintenance in primary cells (37) and to stimulate the BLM helicase through a direct association (38). Recently, we have described the DNA damage hypersensitivity and chromosomal instability phenotypes of Rad51d-deficient cells (9). In the present study, a complementation analysis was developed to rapidly assess the repair capacity of RAD51D mutants by testing polyclonal populations for resistance to MMC. Using this approach, it was determined that mutations in the conserved glycine and lysine residues of Walker Motif A severely limit the ability of RAD51D to repair DNA interstrand crosslinks. Furthermore, yeast two-hybrid studies revealed the inability of these mutants to interact with RAD51C. This study is the first to examine the repair capacity of RAD51D Motif A mutants and to propose a role for ATP binding and hydrolysis in the activation of RAD51D during HR repair.
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Materials and methods |
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Cloning of Rad51c and Xrcc2 cDNA expression constructs
DNA sequence encoding full length Mus musculus Rad51c was obtained by reverse transcribing total RNA from mouse kidney (Superscript; Invitrogen, Carlsbad, CA) and PCR amplified using 51cKpnFor (5'-CTTGGTACCCAGCGGGAGTTGGTGGGT) and 51cBamRev (5'-CAGTTAACTGGATCCACTGGCA) primers. Full-length Mus musculus Xrcc2 was amplified from IMAGE clone 5357630 (American Type Culture Collection, Manassas, VA) using XRCC2For (5'-ATGCTACGGCTCGTGACAGTTCTT) and XRCC2Rev (5'-AGAAGATGACCCTGTGCTTCACGA) primers. Both Rad51c and Xrcc2 amplification products were cloned into a pcDNA3.1/Hygro(+) (KpnI/BamHI) vector encoding an in-frame hemagglutinin (HA) epitope tagging sequence at the 5' end.
Site-directed mutagenesis of Rad51d
A Rad51d cDNA (9) was cloned into the EcoRI and BamHI sites of pUC19 and used as a template for site-directed mutagenesis. To generate Walker Motif A mutants, PCR using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and the following primers sets were used: S111T (5'-GCCCAGGTACCGGCAAAACCCAGGTGTGTCTCTG-3' and 5'CAGAGACACACCTGGGTTTTGCCGGTACCTGGGC-3'), K113R (5'-GCCCAGGTAGCGGCAGAACCCAGGTGTGTCTCTG-3' and 5'-CAGAGACACACCTGGGTTCTGCCGCTACCTGGGC-3'), K113A (5'-GCCCAGGTAGCGGCGCAACCCAGGTGTGTCTCTG-3' and 5'-CAGAGACACACCTGGGTTGCGCCGCTACCTGGGC-3'), G112A (5'-GCCCAGGTAGCGCCAAAACCCAGGTGTGTCTCTG-3' and 5'-CAGAGACACACCTGGGTTTTGGCGCTACCTGGGC-3'), 
G112K113 (5'-GGCCCAGGTAGCACCCAGGTGTGTCTCTGTGTGGCTG-3' and 5'-CAGCCACACAGAGACACACCTGGGTGCTACCTGGGCC-3'). PCR reactions were performed under the following conditions: 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and extension at 68°C for 12 min. Amplified products were digested with DpnI prior to being transformed into ElectroMAX DH12S cells (Invitrogen) by electroporation. Plasmids from ampicillin resistant clones were isolated, and mutants confirmed by DNA sequencing of both strands. The cDNA constructs were subcloned into the KpnI and BamHI restriction sites of a pcDNA3.1/Hygro(+) vector that encodes an in-frame N-terminal influenza HA epitope tag. The cDNA carrying the S111T mutation, which introduced a second KpnI restriction site, was cloned into the NheI and BamHI sites of the pcDNA3.1/Hygro(+) vector and expressed as an HA-tagged fusion protein.
Protein expression and western blotting
One microgram of plasmid constructs were transfected into Rad51d–/– Trp53–/– mouse embryonic fibroblasts (MEFs) using Lipofectamine Plus reagent (Invitrogen). Cells were harvested 48 h post-transfection and suspended in 100 µl of Laemmelli buffer. Subsequently, 30 µl (
100 µg) of each sample was separated on a 10% SDS–PAGE gel and transferred to nitrocellulose membrane. Following blocking for 1 h using 5% dry milk/TBS-T solution (1x Tris buffered saline with 0.1% Tween-20), immunoblotting for HA-tagged proteins was performed by probing with a 1:5000 dilution of anti-HA peroxidase conjugate antibody (3F10; Roche, Indianapolis, IN). Membranes were washed three times in TBS-T, and signals were detected by enhanced chemiluminescence (ECL). Similarly, detection of ß tubulin was performed by probing with a 1:5000 dilution of Anti-ß tubulin (D-10; Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h followed by ECL detection.
Complementation screening
Rad51d–/– Trp53–/– MEFs were grown in monolayer culture in DMEM supplemented with 15% heat inactivated newborn calf serum, L-glutamine and antibiotics as described (9). Plasmid constructs were transfected into Rad51d–/– Trp53–/– MEFs as described above. Twenty-four hours post-transfection cells were trypsinized, mixed and divided equally (7.5 x 105 cells per dish) onto two 150 mm dishes containing regular growth medium. Twenty-four hours after plating, cells were selected either in regular growth medium containing 200 µg/ml hygromycin B with or without the addition of MMC (1 or 4 ng/ml). Colonies were harvested 12 days after selection and fixed with 100% ice-cold methanol prior to Giemsa staining. Plates were coded and colonies containing 
50 cells were scored positive. The percentage of MMC resistant colonies was calculated by dividing the number surviving selection with MMC and hygromycin B by the number that grew in the presence of hygromycin B alone on the duplicate plate.
RT–PCR analysis
RNA was isolated from polyclonal populations (RNAeasy; Qiagen, Valencia, CA), and treated with DNAse prior to reverse transcription using random hexamers and SuperScript III reverse transcriptase according to the manufacturer's instructions (Invitrogen). Amplification of Rad51d cDNA was performed by PCR using primers that bind to sequences from exon 4 (5'-CTACTTGATGCTGGCCTCTATACT) and exon 9 (5'-GGCGACCACTGCAGTGACCGA). PCR conditions were as follows: 35 cycles of denaturation at 94°C for 30 s, annealing at 59°C for 30 s, and extension at 72°C for 1 min. The primer set used to amplify glyceraldehyde 3-phosphate dehydrogenase (G3PDH) was obtained from Clontech (Palo Alto, CA).
Yeast two-hybrid analysis
Rad51d, Rad51c and Xrcc2 inserts containing the N-terminal HA tag were cloned into the EcoRI/BamHI restriction sites of pGADT7 and pGBKT7 yeast two-hybrid vectors (Clontech). Haploid transformants of AH109 and Y187 were generated using the EZ Transformation Kit II (Zymo, Orange, CA). Matings were performed on YPDA plates 16–24 h prior to replica plating onto dropout media. The presence of diploids and genotypes of haploid and mated strains were confirmed by growth on appropriate synthetic dropout media. Colonies plated onto –Ade/–Leu/–His/–Trp media were analyzed for growth throughout a 10 day incubation period at 30°C. Colony lift assays were performed according to the yeast protocols handbook (Clontech). To quantify the protein–protein interactions, liquid ß-galactosidase assays were performed using o-nitrophenyl-ß-D-galactopyranoside as a substrate (ONPG; Sigma, St Louis, MO) as described (39). All ß-galactosidase assays were performed in triplicate with constructs in both the GAL4 activating and DNA binding domains.
Statistical analysis
Statistical significance of the experimental data was determined using SPSS® version 11.0 for Mac OS X. The mean numbers of percentage MMC resistance for each construct and the strength of yeast two-hybrid interactions were compared by ANOVA.
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Complementation screening to measure DNA interstrand crosslink repair
Stable expression of RAD51D was previously demonstrated to correct the DNA interstrand crosslink repair deficiency of Rad51d–/– Trp53–/– mouse embryonic fibroblasts (9
). In the present study, a complementation assay was designed to measure the survival capacity of Rad51d–/– Trp53–/– fibroblasts transfected with a number of mammalian expression constructs. For each experiment, transfected populations were equally divided and either maintained in the presence of growth media containing hygromycin B alone or hygromycin B with addition of the DNA interstrand crosslinking agent MMC as presented in Figure 1A. Percentage resistances were calculated by dividing the number of colonies surviving selection with MMC by those that grew in the presence of hygromycin B alone (Figure 1B). As an initial test of this system, Rad51d-deficient cells were transfected with full-length cDNAs of Rad51d, Rad51c, Xrcc2 or empty vector. In agreement with observations that support non-redundant functions for the RAD51 family members, the percentage resistance of RAD51C and XRCC2 did not differ statistically from vector, and surviving colonies were smaller and stained with less intensity. Approximately 40% of the Rad51d–/– Trp53–/– + Rad51d cDNA fibroblasts within these polyclonal populations are capable of resisting the lethal interstrand crosslinking damage caused by MMC after adjusting for the rate of survival observed for vector alone.
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0.001). Error bars signify the standard error of the mean. Abbreviations: 51D, RAD51D; 51C, RAD51C; X2, XRCC2; Vec, pcDNA3.1/Hygro.Walker Motif A is required for repair of DNA interstrand crosslinks
RAD51D possesses DNA-stimulated ATPase activity consistent with the presence of its ATP binding motifs (40
). To determine the functional significance of Walker Motif A, site-directed mutations in the encoding sequence were generated (Figure 2A). As a positive control, the non-consensus serine at position 111 was replaced by a similar amino acid to introduce a conservative substitution. An S111T mutation was generated because threonine is present at this position in other RAD51 family members (e.g. RAD51 and XRCC2). Expression of each mutant RAD51D protein was confirmed by immunoblotting for the HA tag 48 h post-transfection (Figure 2B).
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The repair activity of each Walker A mutant was tested by complementation screening (Figure 3A). At 1 ng/ml MMC (3 nM), no statistically significant difference was observed between wild-type, mutant and vector transfectants. At 4 ng/ml MMC (12 nM), mutations in the conserved glycine and lysine residues of Walker Motif A reduced the repair capacity of RAD51D ranging from 6- to 23-fold. Approximately 47% of Rad51d–/– Trp53–/– mouse embryonic fibroblasts transfected with the wild-type construct survived selection, and the S111T mutant retained 60% repair activity relative to wild-type (S111T versus WT, P = 0.176). Restoration of MMC resistance by K113R and K113A mutants was 4 and 17% of wild-type levels, respectively. Variation between the K113R and K113A percentage resistances was not statistically significant (K113R versus K113A, P = 0.959). Expression of the G112A mutant resulted in 17% repair activity of the wild-type protein, whereas no ability to resist DNA interstrand crosslinking damage was observed in
G112K113 and vector control populations. Transcript analysis verified expression of all the Rad51d constructs in surviving colonies (Figure 3B). These data confirm that the Walker Motif A mutants cannot restore normal capacity to respond to MMC induced DNA damage in Rad51d–/– Trp53–/– mouse embryonic fibroblasts.
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Walker Motif A mutants display reduced interaction with RAD51C
Recently, it was demonstrated that Walker Motif A mutations in XRCC3 affect the dynamics of RAD51C–XRCC3 interaction (26
). To determine if RAD51D mutants maintain partnerships within the BCDX2 HR repair complex, each mutant was tested for binding with RAD51C and XRCC2 using multiple yeast two-hybrid analyses. A qualitative assessment of ß-galactosidase activity in mated strains is presented in Figure 4. All RAD51D mutants retain the ability to interact with XRCC2; however, binding to RAD51C was diminished as demonstrated by the lack of blue staining in the colony lift assay. The pattern of growth observed on selective media corroborated the reduced capacity of RAD51C to interact with Motif A mutants (Table I). No growth was observed for haploids or vector control matings confirming the specificity of these interactions.
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To quantify the loss of binding between RAD51D mutants and RAD51C, ß-galactosidase activity was measured using o-nitrophenyl-ß-D-galactopyranoside as a substrate. In agreement with results obtained from colony lift assays, Walker Motif A mutants display a reduced ability to interact with RAD51C (Figure 5A). Each of the Walker Motif A mutations targeting the conserved glycine and lysine residues displayed an
8-fold reduction in RAD51C binding ability, whereas the S111T interaction was similar to wild-type (S111T versus WT, P = 0.926). The average strength of the K113R–XRCC2 and K113A–XRCC2 interactions were maintained at 57 and 54% of wild-type levels, respectively, with some small variation dependent upon orientation of the GAL4 fusion. Both the G112A and deletion mutant retained an ability to interact with XRCC2 at 18% of the wild-type level. These data suggest that conservation of the Walker Motif A in RAD51D is required for interaction with RAD51C; however, binding to XRCC2 occurs in the presence of both conservative and non-conservative substitutions of lysine 113 (K113R versus S111T, P = 0.959; K113A versus S111T, P = 1.00).
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These cell-based studies are the first evidence that conservation of Walker Motif A is required for a physiological role of RAD51D. Each mutation in Walker Motif A, predicted to reduce ATPase activity, severely affected resistance to DNA interstrand crosslinks. Furthermore, these same mutants lacked the capacity to associate with the known RAD51D binding partner RAD51C. We therefore propose a requirement for RAD51D ATP binding and hydrolysis during the repair of DNA interstrand crosslinks (Figure 6). Based upon studies of structurally similar proteins that contain Walker Motifs, both K113R and K113A are expected to lack the ability to hydrolyze ATP whereas the K113R mutant is predicted to allow as much as 40% of the wild-type ATP binding activity (19
). Because the RAD51D K113R mutant did not complement the sensitivity to DNA interstrand crosslinks, an ability to bind ATP is not sufficient for repair. On the contrary, binding and subsequent hydrolysis of ATP by RAD51D is necessary for its association with RAD51C and DNA interstrand crosslink repair. Complexes dependent upon a direct interaction between RAD51D and RAD51C would also be affected by the inability of RAD51D to hydrolyze ATP. Thus, the data presented in Figure 3 support the conclusion that a recombinosome containing both RAD51C and RAD51D (e.g. the BCDX2 complex) is required for at least one step during the restoration of DNA interstrand crosslinks.
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Our data imply that ATP hydrolysis results in a conformational change in RAD51D that favors interaction with RAD51C (illustrated in Figure 6 by the transition from hexagon to sphere in the C-terminal domain of RAD51D). The ability of K113R and K113A mutants to interact with XRCC2 suggests the allosteric effect specifically regulates the binding of RAD51D to RAD51C. Although results from the ONPG assay suggest a lower level of interaction between XRCC2 and G112A/
G112K113 mutants, binding was detectable using techniques with increased sensitivity (Figure 4 and Table I). The reduced interaction may reflect a destabilization of the protein resulting from the severity of these non-conservative amino acid substitutions. However, it is unlikely that the absence of binding of the Motif A mutants to RAD51C results from lack of protein expression because (i) multiple matings using independently purified single colonies generated identical results and (ii) isogenic clones displayed growth when mated with strains expressing XRCC2. It has been demonstrated that stability of the RAD51C and XRCC3 proteins is dependent upon their interaction (41,42). It is possible that failure of the RAD51D Motif A mutants to complement the MMC sensitivity phenotype is in part the result of destabilization of RAD51C or RAD51D if the two proteins fail to interact. Nevertheless, these protein interaction experiments were conducted in yeast, and it remains possible that the outcomes do not exactly reflect the mammalian interactions in vivo. Because the BCDX2 recombinosome demonstrates greater ATPase activity than RAD51 or RAD51D alone (40,43), it follows that other members of the complex (e.g. RAD51B and RAD51C) may contribute additive ATP hydrolysis required for efficient interstrand crosslink repair.
Unlike the model proposed by Yamada et al. (26) in which ATP binding disrupts the RAD51C–XRCC3 complex, yeast two-hybrid analyses demonstrate that conservation of lysine at position 113 in RAD51D is a requirement for its partnership with RAD51C. Therefore, ATP binding and hydrolysis appear to favor the RAD51C–RAD51D interaction whereas ATP binding facilitates disassociation of the RAD51C–XRCC3 heterodimer. A recent computational model based upon the crystal structure of archaeal RadA is in agreement with published experimental analyses of the pairwise interactions between the RAD51 paralogs with the exception of that proposed for RAD51C–XRCC3 (44). Therefore, it is conceivable that RAD51C–XRCC3 displays a binding modality that is unique from the interaction observed between RAD51C and RAD51D within the BCDX2 recombinosome. With this presumption in mind, the notion that ATPase activity has opposite regulatory effects on the dynamics of RAD51C–XRCC3 and RAD51C–RAD51D interactions is currently an attractive hypothesis.
The data presented here and the recent study by Yamada et al. (26) regarding the dynamics of the RAD51C–XRCC3 complex raise the interesting possibility that the RAD51 paralogs may serve as molecular switches during HR repair of DNA interstrand crosslinks. We speculate paralog complexes could possess functions analogous to the MutS homologs of DNA mismatch repair. The ability of these proteins to hydrolyze ATP is critical for their normal function, and mutation of conserved amino acid residues in the ATPase domain results in dominant mutator or dominant negative phenotypes (45–47). During the mismatch repair process, the hMSH2–hMSH6 heterodimer functions as an ATPase switch that regulates mismatch binding (48). The presence of such ‘switch regions’ that facilitate conformational changes in RecA and ABC transport proteins have been reported (49); however, a homologous region in RAD51D has not been identified.
Mutations of the ATPase motif found in four of the five RAD51 paralogs have now been characterized. Walker Motif A in RAD51C and XRCC3 is required for the repair of DNA interstrand crosslinks (25,26); however, conservation of Motif A does not appear to be necessary for the involvement of XRCC2 in HR (29). Because mutagenesis of RAD51D suggests a functional requirement for ATPase activity, detailed biochemical analyses of these mutants are now needed to determine precisely how ATP binding, hydrolysis and turnover affect its role in HR repair. It remains to be determined whether wild-type cells expressing these mutants will be hypersensitive to DNA interstrand crosslinks (e.g. a dominant negative phenotype) or display telomere dysfunction. In addition, the capability of the Walker Motif A mutants to maintain interaction with the BLM helicase is also unknown.
In conclusion, the ATPase motif in RAD51D is required for DNA interstrand crosslink repair and interaction with RAD51C. For the first time, we have assessed the functional requirement of conserved residues in RAD51D. Because cell lines defective for other RAD51 paralogs display hypersensitivity to DNA interstrand crosslinks, the complementation strategy presented here could be used to rapidly identify mutations in Rad51 related genes and examine the functional activity of paralog isoforms (50–52). In addition, known polymorphisms in Rad51d that are associated with increased cancer risk can now be examined (16). The results presented here contribute towards a mechanistic understanding of DNA interstrand crosslink repair and provide insight on the role RAD51 paralogs have in cancer prevention through repair of these lesions.
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Acknowledgments |
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We thank Kandace Williams, Jean Overmeyer and Aditi Nadkarni for critical reading of the manuscript. This work was supported by an American Cancer Society grant to D.L.P. (RSG-030158-01-GMC). A.M.G. was supported by an MD/PhD predoctoral fellowship from the Medical University of Ohio. Financial support was given by The American Cancer Society.
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* To whom correspondence should be addressed at Department of Physiology and Cardiovascular Genomics, Medical University of Ohio, Block Health Science Building, 3035 Arlington Avenue, Toledo, OH 43614-5804, USA. Tel: +1 419 383 4370; Fax: +1 419 383 6168; Email: dpittman{at}meduohio.edu.
2 Present address: Genentech, Inc., South San Francisco, CA, USA 
3 Present address: University of California, Davis, Sacramento, CA, USA
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1. Wyman,C., Ristic,D. and Kanaar,R. (2004) Homologous recombination-mediated double-strand break repair. DNA Repair (Amst.), 3, 827–833.[CrossRef][Medline]
2. Pfeiffer,P., Goedecke,W., Kuhfittig-Kulle,S. and Obe,G. (2004) Pathways of DNA double-strand break repair and their impact on the prevention and formation of chromosomal aberrations. Cytogenet Genome Res., 104, 7–13.[CrossRef][ISI][Medline]
3. Griffin,C.S. and Thacker,J. (2004) The role of homologous recombination repair in the formation of chromosome aberrations. Cytogenet Genome Res., 104, 21–27.[Medline]
4. Stark,J.M. and Jasin,M. (2003) Extensive loss of heterozygosity is suppressed during homologous repair of chromosomal breaks. Mol. Cell. Biol., 23, 733–743.
5. Masson,J.Y., Tarsounas,M.C., Stasiak,A.Z., Stasiak,A., Shah,R., McIlwraith,M.J., Benson,F.E. and West,S.C. (2001) Identification and purification of two distinct complexes containing the five RAD51 paralogs. Genes Dev., 15, 3296–3307.
6. Liu,N., Schild,D., Thelen,M.P. and Thompson,L.H. (2002) Involvement of Rad51C in two distinct protein complexes of Rad51 paralogs in human cells. Nucleic Acids Res., 30, 1009–1015.
7. Miller,K.A., Yoshikawa,D.M., McConnell,I.R., Clark,R., Schild,D. and Albala,J.S. (2002) RAD51C interacts with RAD51B and is central to a larger protein complex in vivo exclusive of RAD51. J. Biol. Chem., 277, 8406–8411.
8. Wiese,C., Collins,D.W., Albala,J.S., Thompson,L.H., Kronenberg,A. and Schild,D. (2002) Interactions involving the Rad51 paralogs Rad51C and XRCC3 in human cells. Nucleic Acids Res., 30, 1001–1008.
9. Smiraldo,P.G., Gruver,A.M., Osborn,J.C. and Pittman,D.L. (2005) Extensive chromosomal instability in Rad51d-deficient mouse cells. Cancer Res., 65, 2089–2096.
10. Deans,B., Griffin,C.S., O'Regan,P., Jasin,M. and Thacker,J. (2003) Homologous recombination deficiency leads to profound genetic instability in cells derived from Xrcc2-knockout mice. Cancer Res., 63, 8181–8187.
11. Takata,M., Sasaki,M.S., Tachiiri,S., Fukushima,T., Sonoda,E., Schild,D., Thompson,L.H. and Takeda,S. (2001) Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Mol. Cell. Biol., 21, 2858–2866.
12. Liu,N., Lamerdin,J.E., Tebbs,R.S. et al. (1998) XRCC2 and XRCC3, new human Rad51-family members, promote chromosome stability and protect against DNA cross-links and other damages. Mol. Cell, 1, 783–793.[CrossRef][ISI][Medline]
13. Pittman,D.L., Cobb,J., Schimenti,K.J., Wilson,L.A., Cooper,D.M., Brignull,E., Handel,M.A. and Schimenti,J.C. (1998) Meiotic prophase arrest with failure of chromosome synapsis in mice deficient for Dmc1, a germline-specific RecA homolog. Mol. Cell, 1, 697–705.[CrossRef][ISI][Medline]
14. Thacker,J. (2005) The RAD51 gene family, genetic instability and cancer. Cancer Lett., 219, 125–135.[CrossRef][ISI][Medline]
15. Richardson,C. (2005) RAD51, genomic stability, and tumorigenesis. Cancer Lett., 218, 127–139.[CrossRef][ISI][Medline]
16. Rodriguez-Lopez,R., Osorio,A., Ribas,G. et al. (2004) The variant E233G of the RAD51D gene could be a low-penetrance allele in high-risk breast cancer families without BRCA1/2 mutations. Int. J. Cancer, 110, 845–849.[CrossRef][ISI][Medline]
17. Shivji,M.K. and Venkitaraman,A.R. (2004) DNA recombination, chromosomal stability and carcinogenesis: insights into the role of BRCA2. DNA Repair (Amst.), 3, 835–843.[CrossRef][Medline]
18. Walker,J.E., Saraste,M., Runswick,M.J. and Gay,N.J. (1982) Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J., 1, 945–951.[ISI][Medline]
19. Sung,P., Higgins,D., Prakash,L. and Prakash,S. (1988) Mutation of lysine-48 to arginine in the yeast RAD3 protein abolishes its ATPase and DNA helicase activities but not the ability to bind ATP. EMBO J., 7, 3263–3269.[ISI][Medline]
20. Story,R.M. and Steitz,T.A. (1992) Structure of the recA protein–ADP complex. Nature, 355, 374–376.[CrossRef][Medline]
21. Sung,P. and Stratton,S.A. (1996) Yeast Rad51 recombinase mediates polar DNA strand exchange in the absence of ATP hydrolysis. J. Biol. Chem., 271, 27983–27986.
22. Konola,J.T., Logan,K.M. and Knight,K.L. (1994) Functional characterization of residues in the P-loop motif of the RecA protein ATP binding site. J. Mol. Biol., 237, 20–34.[CrossRef][ISI][Medline]
23. Rehrauer,W.M. and Kowalczykowski,S.C. (1993) Alteration of the nucleoside triphosphate (NTP) catalytic domain within Escherichia coli recA protein attenuates NTP hydrolysis but not joint molecule formation. J. Biol. Chem., 268, 1292–1297.
24. Stark,J.M., Hu,P., Pierce,A.J., Moynahan,M.E., Ellis,N. and Jasin,M. (2002) ATP hydrolysis by mammalian RAD51 has a key role during homology-directed DNA repair. J. Biol. Chem., 277, 20185–20194.
25. French,C.A., Tambini,C.E. and Thacker,J. (2003) Identification of functional domains in the RAD51L2 (RAD51C) protein and its requirement for gene conversion. J. Biol. Chem., 278, 45445–45450.
26. Yamada,N.A., Hinz,J.M., Kopf,V.L., Segalle,K.D. and Thompson,L.H. (2004) XRCC3 ATPase activity is required for normal XRCC3–Rad51C complex dynamics and homologous recombination. J. Biol. Chem., 279, 23250–23254.
27. Rafii,S., Lindblom,A., Reed,M., Meuth,M. and Cox,A. (2003) A naturally occurring mutation in an ATP-binding domain of the recombination repair gene XRCC3 ablates its function without causing cancer susceptibility. Hum. Mol. Genet., 12, 915–923.
28. Johnson,R.D. and Symington,L.S. (1995) Functional differences and interactions among the putative RecA homologs Rad51, Rad55, and Rad57. Mol. Cell. Biol., 15, 4843–4850.[Abstract]
29. O'Regan,P., Wilson,C., Townsend,S. and Thacker,J. (2001) XRCC2 is a nuclear RAD51-like protein required for damage-dependent RAD51 focus formation without the need for ATP binding. J. Biol. Chem., 276, 22148–22153.
30. Lio,Y.C., Mazin,A.V., Kowalczykowski,S.C. and Chen,D.J. (2003) Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro. J. Biol. Chem., 278, 2469–2478.
31. Shin,D.S., Chahwan,C., Huffman,J.L. and Tainer,J.A. (2004) Structure and function of the double-strand break repair machinery. DNA Repair (Amst.), 3, 863–873.[Medline]
32. Bork,J.M., Cox,M.M. and Inman,R.B. (2001) RecA protein filaments disassemble in the 5' to 3' direction on single-stranded DNA. J. Biol. Chem., 276, 45740–45743.
33. Menetski,J.P. and Kowalczykowski,S.C. (1985) Interaction of recA protein with single-stranded DNA. Quantitative aspects of binding affinity modulation by nucleotide cofactors. J. Biol. Chem., 181, 281–295.
34. Namsaraev,E.A. and Berg,P. (1998) Interaction of Rad51 with ATP and Mg2+ induces a conformational change in Rad51. Biochemistry, 37, 11932–11939.[CrossRef][Medline]
35. Kim,H.K., Morimatsu,K., Norden,B., Ardhammar,M. and Takahashi,M. (2002) ADP stabilizes the human Rad51-single stranded DNA complex and promotes its DNA annealing activity. Genes Cells, 7, 1125–1134.[Abstract]
36. Kurumizaka,H., Ikawa,S., Nakada,M., Enomoto,R., Kagawa,W., Kinebuchi,T., Yamazoe,M., Yokoyama,S. and Shibata,T. (2002) Homologous pairing and ring and filament structure formation activities of the human Xrcc2*Rad51D complex. J. Biol. Chem., 277, 14315–14320.
37. Tarsounas,M., Munoz,P., Claas,A., Smiraldo,P.G., Pittman,D.L., Blasco,M.A. and West,S.C. (2004) Telomere maintenance requires the RAD51D recombination/repair protein. Cell, 117, 337–347.[CrossRef][ISI][Medline]
38. Braybrooke,J.P., Li,J.L., Wu,L., Caple,F., Benson,F.E. and Hickson,I.D. (2003) Functional interaction between the Bloom's syndrome helicase and the RAD51 paralog, RAD51L3 (RAD51D). J. Biol. Chem., 278, 48357–48366.
39. Miller,K.A., Sawicka,D., Barsky,D. and Albala,J.S. (2004) Domain mapping of the Rad51 paralog protein complexes. Nucleic Acids Res., 32, 169–178.
40. Braybrooke,J.P., Spink,K.G., Thacker,J. and Hickson,I.D. (2000) The RAD51 family member, RAD51L3, is a DNA-stimulated ATPase that forms a complex with XRCC2. J. Biol. Chem., 275, 29100–29106.
41. Lio,Y.C., Schild,D., Brenneman,M.A., Redpath,J.L. and Chen,D.J. (2004) Human Rad51C deficiency destabilizes XRCC3, impairs recombination, and radiosensitizes S/G2-phase cells. J. Biol. Chem., 279, 42313–42320.
42. Xu,Z.Y., Loignon,M., Han,F.Y., Panasci,L. and Aloyz,R. (2005) Xrcc3 induces Cisplatin resistance by stimulation of rad51-related recombinational repair, S-phase checkpoint activation, and reduced apoptosis. J. Pharmacol. Exp. Ther., 314, 495–505.
43. Yokoyama,H., Sarai,N., Kagawa,W., Enomoto,R., Shibata,T., Kurumizaka,H. and Yokoyama,S. (2004) Preferential binding to branched DNA strands and strand-annealing activity of the human Rad51B, Rad51C, Rad51D and Xrcc2 protein complex. Nucleic Acids Res., 32, 2556–2565.
44. Ariza,A., Richard,D.J., White,M.F. and Bond,C.S. (2005) Conformational flexibility revealed by the crystal structure of a crenarchaeal RadA. Nucleic Acids Res., 33, 1465–1473.
45. Haber,L.T. and Walker,G.C. (1991) Altering the conserved nucleotide binding motif in the Salmonella typhimurium MutS mismatch repair protein affects both its ATPase and mismatch binding activities. EMBO J., 10, 2707–2715.[ISI][Medline]
46. Wu,T.H. and Marinus,M.G. (1994) Dominant negative mutator mutations in the mutS gene of Escherichia coli. J. Bacteriol., 176, 5393–5400.
47. Alani,E., Sokolsky,T., Studamire,B., Miret,J.J. and Lahue,R.S. (1997) Genetic and biochemical analysis of Msh2p–Msh6p: role of ATP hydrolysis and Msh2p–Msh6p subunit interactions in mismatch base pair recognition. Mol. Cell. Biol., 17, 2436–2447.[Abstract]
48. Gradia,S., Acharya,S. and Fishel,R. (1997) The human mismatch recognition complex hMSH2-hMSH6 functions as a novel molecular switch. Cell, 91, 995–1005.[CrossRef][ISI][Medline]
49. Schneider,E. and Hunke,S. (1998) ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains. FEMS Microbiol. Rev., 22, 1–20.[CrossRef][ISI][Medline]
50. Schoenmakers,E.F., Huysmans,C. and Van de Ven,W.J. (1999) Allelic knockout of novel splice variants of human recombination repair gene RAD51B in t(12;14) uterine leiomyomas. Cancer Res., 59, 19–23.
51. Kawabata,M., Akiyama,K. and Kawabata,T. (2004) Genomic structure and multiple alternative transcripts of the mouse TRAD/RAD51L3/RAD51D gene, a member of the recA/RAD51 gene family. Biochim. Biophys. Acta, 1679, 107–116.[Medline]
52. Kawabata,M. and Saeki,K. (1999) Multiple alternative transcripts of the human homologue of the mouse TRAD/R51H3/RAD51D gene, a member of the rec A/RAD51 gene family. Biochem. Biophys. Res. Commun., 257, 156–162.[CrossRef][ISI][Medline]
53. Pittman,D.L., Weinberg,L.R. and Schimenti,J.C. (1998) Identification, characterization, and genetic mapping of Rad51d, a new mouse and human RAD51/RecA-related gene. Genomics, 49, 103–111.[CrossRef][ISI][Medline]
54. Pittman,D.L. and Schimenti,J.C. (2000) Midgestation lethality in mice deficient for the RecA-related gene, Rad51d/Rad51l3. Genesis, 26, 167–173.[CrossRef][ISI][Medline]
55. Liu,Y., Masson,J.Y., Shah,R., O'Regan,P. and West,S.C. (2004) RAD51C is required for Holliday junction processing in mammalian cells. Science, 303, 243–246.
Received on May 2, 2005; revised on August 27, 2005; accepted on September 27, 2005.
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