Monday, January 13, 2025
1/13/2025
PROJECT SUMMARY/ABSTRACT Homologous recombination is an essential DNA repair process that facilitates accurate chromosome segregation during meiosis, the specialized cell division that produces sperm and eggs. A fundamental gap in our understanding of recombination during meiosis in mammals is the role the breast cancer associated protein, BRCA2, which loads the central recombination enzyme RAD51 onto the ends of DNA breaks. This knowledge gap persists because essential factors like BRCA2 are inherently hard to study, and reagents to detect and analyze BRCA2 are limiting. These impediments are overcome in this proposal with the development of genetic, genomic, and biochemical reagents and approaches to study the meiotic functions of BRCA2. These include a fully functional epitope-tagged allele of the native Brca2 gene in mouse; high- resolution mapping approaches to define BRCA2 binding sites genome wide; germ-cell specific Cre-lox mouse lines to ablate Brca2 gene function at different stages of meiosis; and purified full-length human BRCA2 protein and its partner proteins for biochemical analysis. Exploiting this unique constellation of tools, the long-term objective of this project, to understand the roles and regulation of BRCA2 during meiosis in mammals, will be pursued through three aims. Aim 1 will define the spatial-temporal binding patterns of BRCA2 and related recombination factors using super-resolution immunofluorescence imaging, complemented by genome-wide ChIP-Seq mapping. Aim 2 will determine the roles of BRCA2 throughout meiosis by inactivating the Brca2 gene in mouse spermatocytes and oocytes before, during and after meiotic prophase. Mutant phenotypes will be analyzed using a battery physiological, histological, cell biology, and molecular assays. Preliminary data support the hypothesis that BRCA2 functions at multiple stages of meiosis, and in the maintenance of ovarian reserves. Aim 3 will define how a meiosis-specific chromosomal protein, SYCP3, modulates meiotic functions of BRCA2 to optimize meiotic HR. Super-resolution imaging and genome-wide ChIP-Seq mapping of BRCA2 and related recombination factors will be performed in Sycp3 mutant mice. Biochemical assays with purified human proteins will test the hypothesis that SYCP3 binds BRCA2 to facilitate the balanced assembly of RAD51 and its meiotic homolog DMC1 at recombination sites to promote DMC1-catalyzed recombination between homologous chromosomes. The results of these aims will provide unprecedented insights into the missing biology of BRCA2 function during meiosis in mammals. These fundamental discoveries will be germane to understanding pathologies associated with human meiosis, including infertility, miscarriage, congenital disease, and premature ovarian insufficiency.
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