Title Tumor-associated mutations in a conserved structural motif alter physical and biochemical properties of human RAD 51 recombinase Permalink

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. INTRODUCTION Human RAD51 protein is a RecA-like DNA strand transferase that catalyzes the central reactions of homologous pairing and DNA strand exchange in the homologous recombination (HR) and homology-directed repair (HDR) pathways. Given the crucial roles played by HR in distinctive cellular genome stability processes, a linkage between HR dysfunction and cancer phenotype is now clear and widely accepted (1). The causal relationship between RAD51 abnormalities and cancer, however, is poorly understood. Elevated expression of RAD51 protein has been observed in a wide variety of cancers suggesting that overexpression of RAD51 confers advantages to tumor cells (2). Increased RAD51 expression was found in invasive ductal breast cancer and the level of overexpression correlated directly with the histological grading of the tumor (3). As HR machinery components have now become crucial targets for cancer treatment, there is a widely observed correlation between expression levels of RAD51 and resistance to therapeutic drugs (4). High RAD51 expression was also reported in malignant prostate and small cell lung cancers (5). Conversely, the expression of RAD51, as well as BRCA1, is reduced in almost one third of breast tumor cell lines and primary sporadic breast cancer cells (6–9). Consistent with the drug resistance of the cancer cells conferred by the overexpression of RAD51, down-regulation of RAD51 also increased the radio-sensitivity of prostate cancer cells (10) and malignant glioma cells (11). Although the mechanism and significance of such deregulation of RAD51 remains speculative, a basal level of RAD51 activity appears to be kept in an elaborate balance to ensure appropriate functionality of RAD51-dependent HR in specific contexts. *To whom correspondence should be addressed. Tel: +1 802 656 8260; Fax: +1 802 656 8220; Email: smorrica@uvm.edu Present address: Jianhong Chen, Laboratory of Biochemistry and Molecular Biology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892,