Repair of DNA interstrand crosslinks requires action of multiple DNA repair pathways, including homologous recombination. Here, we report a de novo heterozygous T131P mutation in RAD51/FANCR, the key recombinase essential for homologous recombination, in a patient with Fanconi anemia-like phenotype. Invitro, RAD51-T131P displays DNA-independent ATPase activity, no DNA pairing capacity, and a co-dominant-negative effect on RAD51 recombinase function. However, the patient cells are homologous recombination proficient due to the low ratio ofmutantto wild-type RAD51 in cells. Instead, patientcells aresensitive to crosslinking agents and display hyperphosphorylation of Replication Protein A due to increased activity of DNA2 and WRN at the DNA interstrand crosslinks. Thus, proper RAD51 function is important during DNA interstrand crosslink repair outside of homologous recombination. Our study provides a molecular basis for how RAD51 and its associated factors may operate in a homologous recombination-independent manner to maintain genomic integrity.
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We thank the patient and her family, without whom this study would not be possible. We thank Steve West and John Petrini for providing us with anti-RAD51 and anti-MRE11 antibodies, respectively; Nikola Pavletich for the RAD51 cDNA construct; and Detlev Schindler for sharing the RAD50 mutant cell line. We are grateful to all members of the A.S. laboratory for helpful discussion and critical comments on the manuscript. This work was supported by a Starr Center Consortium grant (A.S. and S.B.G.), the Rita Allen Foundation and Burroughs Wellcome Foundation (A.S.), an Irma T. Hirschl Research Award (A.S.), a Doris Duke Clinical Scientist Development Award (A.S.), NIH grant GM62653 (S.C.K.), grant #8 UL1 TR000043 from the National Center for Advancing Translational Sciences (NCATS), and the NIH Clinical and Translational Science Award (CTSA) program. The IFAR is partially supported by NIH grant R01HL120922 (A.S.). A.T.W. was partially funded by Congressionally Directed Medical Research Programs and a Bone Marrow Failure Research Program Postdoctoral Fellowship Training Award (grant log #BM120004).
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