Robust cellular repair pathways maintain genomic integrity by eliminating or reducing genetic alterations following DMA damage. In the absence or malfunction of such pathways, health is compromised and individuals can be cancer-prone. Double-strand breaks (DSBs) are a particularly injurious type of DMA damage. DSBs occur spontaneously in chromosomes as a result of normal DMA metabolism and after exposure to exogenous DNA damaging agents, such as ionizing radiation. We have demonstrated that a major pathway involved in DSB repair is homologous recombination (HR). In HR, an undamaged homologous sequence templates the repair of the broken chromosome, making it a precise type of repair. The proposed research focuses on understanding the factors that promote HR in mammalian cells and the consequences of impaired HR on organismal health.
Specific aim 1 is a structure-function analysis of the tumor suppressor BRCA2 and its role in promoting cell survival, HR, and organismal viability in the mouse. There is a specific emphasis on the role of various subdomains of the BRCA2 DNA binding domain. In addition, the role of several patient mutations will be investigated.
In specific aim 2 the cellular requirements for HR correction in Brca2 and other HR mutant cells will be investigated. Our preliminary analysis indicates that the function of BRCA2 in HR is to deliver the Rad51 strand exchange protein to single-stranded DNA. Experiments are proposed to test and extend this hypothesis are proposed.
Specific aim 3 is to investigate the dependence on HR factors in the mouse. Mice with severe HR deficiency die early in embryogenesis, although mice with less severe deficiencies progress further in development or are viable. To understand the dependencies and uncover roles for HR factors in the mouse, we will analyze mice that carry mutations in combinations of HR factors. The roles of a gene encoding a Rad51 paralog (Xrcc2) and other Rad52 epistasis group members (Rad52 and Rad54) will be investigated, as well as that of a hypomorphic Brca2 allele.
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