Structural Analysis of Cancer-Relevant BCRA2 Mutations
Creators: name: University Of California Office Of The President
Publisher: name: University of California Office of the President
publisherIdentifier: https://ror.org/00dmfq477
publisherIdentifierScheme: ROR
Year: 2024

description: Inherited mutations in human BRCA1 and BRCA2 are responsible for about 5-10% of the total breast cancer cases and about one-half of all familial cases of breast and ovarian cancer. While evidence for a role of BRCA2 in the recombinational repair of DNA damage is mounting, the precise molecular function(s) of this protein and its biochemical properties remain unknown. Rad51 is the central protein in recombinational repair that performs the critical steps of homology search and DNA strand exchange in the repair of double-stranded DNA breaks. The biochemistry and function of Rad51 protein is comparably well understood. The interaction between BRCA2 and Rad51 appears to be critical for the cellular function of BRCA2 protein. Over 100 different mutations are known for BRCA2, which can be both, truncation mutations (i.e. shortened protein) and point mutations (i.e., amino acid sequence changes). 
 
 
 
 
 
 
 
 We wish to determine the three-dimensional (3D) structure of truncation and substitution mutants and to test the hypothesis that a subgroup of point mutations results in a folded BRCA2 protein. These changes are likely to result in a reduced ability to bind to Rad51, thereby inferring the elevated cancer risk. Human BRCA2 is an exceptionally large protein with 3,418 amino acids. In the foreseeable future it is highly unlikely that sufficient amounts of full-length protein will become available to undertake traditional biochemical studies. However, analysis by cryo-electron microscopy (i.e., a process of imaging a frozen sample, called cryoEM) is now an attainable goal. The objective of this proposal is to apply cryoEM to the structural and functional analysis of human BRCA2 with breast cancer-relevant mutations. This effort is a collaboration between two laboratories with complementary expertise in the biochemistry of DNA binding proteins and in cryoEM. We have purified human Rad51, and have established a protocol to purify small amounts full-length human BRCA2 protein. Applying the same methods, we will produce BRCA2 protein with breast-cancer relevant mutations, and analyze their structure and Rad51 binding behavior. Frozen hydrated cryoEM samples will be imaged using a transmission electron microscope (TEM). Computer image processing will be used to generate a ”library” of 3D structures of the different mutant BRCA2 proteins, with and without Rad51. Our hypothesis is that a subgroup of such mutant BRCA2 proteins may become candidates for mutation-specific drug development efforts.
 
 
 
 
 
 
 
 The structural and protein binding properties of BRCA2 protein is still largely unknown, and its precise cellular and molecular role is not understood. Comparing the 3-D structures of BRCA2 and/or BRCA2-Rad51 complexes with different breast-cancer relevant mutations will provide a structural basis to identify the cellular and molecular functions of this important tumor suppressor protein. This will be an essential milestone on the critical path to structure-assisted drug design, even though this is a very early step.