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The breast cancer suppressor BRCA2 controls the recombinase RAD51 in the reactions that mediate homologous DNA recombination, an essential cellular process required for the error-free repair of DNA double-stranded breaks. The primary mode of interaction between BRCA2 and RAD51 is through the BRC repeats, which are ∼35 residue peptide motifs that interact directly with RAD51 in vitro. Human BRCA2, like its mammalian orthologues, contains 8 BRC repeats whose sequence and spacing are evolutionarily conserved. Despite their sequence conservation, there is evidence that the different human BRC repeats have distinct capacities to bind RAD51. A previously published crystal structure reports the structural basis of the interaction between human BRC4 and the catalytic core domain of RAD51. However, no structural information is available regarding the binding of the remaining seven BRC repeats to RAD51, nor is it known why the BRC repeats show marked variation in binding affinity to RAD51 despite only subtle sequence variation. To address these issues, we have performed fluorescence polarisation assays to indirectly measure relative binding affinity, and applied computational simulations to interrogate the behaviour of the eight human BRC-RAD51 complexes, as well as a suite of BRC cancer-associated mutations. Our computational approaches encompass a range of techniques designed to link sequence variation with binding free energy. They include MM-PBSA and thermodynamic integration, which are based on classical force fields, and a recently developed approach to computing binding free energies from large-scale quantum mechanical first principles calculations with the linear-scaling density functional code onetep. Our findings not only reveal how sequence variation in the BRC repeats directly affects affinity with RAD51 and provide significant new insights into the control of RAD51 by human BRCA2, but also exemplify a palette of computational and experimental tools for the analysis of protein-protein interactions for chemical biology and molecular therapeutics.
Pubmed ID: 21789034 RIS Download
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When the BRCA1 gene was cloned, a Steering Committee was initiated to help coordinate the formation of a Breast Cancer Information Core (BIC) that could act as such a central repository. NHGRI has chosen as the most accessible format for the BIC this World Wide Web site. The recent identification of mutations in breast cancer susceptibility genes has provided the exciting opportunity to help identify women who are at high risk to develop breast cancer. One of the serious impediments to achieving clinical benefits from this information however, is finding and assessing the significance of mutations in these new susceptibility genes. It is imperative that the detection and interpretation of these mutations is coordinated and that this information is made available to as many qualified investigators as possible. There are many sites on the web that contain general as well as scientific information relevant to breast cancer. A partial list of these can be found here. Having participated in the poorly coordinated analysis of other cancer susceptibility genes, we consider it important to create and maintain a central repository for information regarding mutations and polymorphisms. NHGRI also think it critical to make available the reagents necessary to carry out many different techniques for the detection of such mutations. Sponsors: This resource is supported by the National Human Genome Research Institute (NHGRI). Keywords: Breast, Cancer, Mutation, Clincial, Polymorphism, Gene, Scientific,
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