My lab has a long-standing interest in mechanisms and factors involved in homologous recombination (HR) in mammalian cells. We established that HR is a major repair mechanism in mammalian cells for DNA double-strand breaks (DSBs) and that breast and ovarian tumor suppressors are HR factors. In addition, we demonstrated with my colleague Scott Keeney that the mouse SPO11 protein introduces DSBs to initiate meiotic HR and that the non-Mendelian transfer of information - gene conversion - occurs during meiotic HR in the mouse. The importance of HR in somatic cells is particularly emphasized by the association of mutations in HR genes with human disease, especially cancer, while in germ cells, mistakes in HR can lead to miscarriage and developmental defects. The application builds on our earlier discoveries with the long-term goals of understanding HR mechanisms and factors in mitotic and meiotic cells. Our previous observations demonstrated that meiotic gene conversion occurs more broadly through meiotic HR hotspots than expected from relatively focused DSB formation, which has implications for the evolutionary longevity of hotspots. We will explore possible mechanisms that could lead to this pattern of gene conversion, focusing on the role of mismatch repair factors, as well as how sequence divergence affects HR frequency and outcomes. While HR between homologous chromosomes is critical in meiotic cells, it can be deleterious in mitotic cells if it leads to loss of heterozgosity. However, studies of mitotic interhomolog recombination have lagged behind those in meiotic cells. We will determine how mechanisms of meiotic and mitotic interhomolog HR compare and how the different cellular contexts impact DNA transactions. During meiosis, DSB numbers as well as position need to be tightly regulated. The ATM kinase plays a critical role in regulating meiotic DSB numbers; we will address whether ATM also controls the spatial regulation of DSB formation, including on the pseudoautosomal region shared by the sex chromosomes. Proper DSB repair by HR is critical in meiosis, as typically one to two crossovers occur on each homolog, with much of the remaining repair presumed to give rise to interhomolog noncrossovers. We will address whether loss of ATM leads to aberrant DSB repair outcomes. Many HR factors are crucial in mammals as their loss can lead to embryonic lethality, developmental defects, sterility, or tumorigenesis. We have begun to explore the function of additional presumptive HR factors and plan an integrated approach to understand the role of these factors in the animal in relation to that of known HR factors. Finally, the choice for a DSB to undergo HR or more mutagenic nonhomologous repair appears to be controlled at the initial end processing step, termed end resection. However, this process and its control are poorly understood in mammalian cells. We will develop nucleotide resolution end resection analysis to gain insight into this key control step.
Lesions that arise in the genome compromise its integrity and so must be repaired, since lack of repair or misrepair leads to genomic loss or rearrangements, which are associated with many tumor types, including breast and ovarian cancer. Conversely, some lesions are beneficial because their repair leads to proper gamete formation by promoting the segregation of maternal and paternal chromosomes, errors of which can lead to developmental issues. This project addresses fundamental questions about the repair of DNA lesions in which both strands of DNA are broken, and impacts our human fertility, development, and cancer.
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