Molecular Mechanisms of Human Homologous Recombination

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SUMMARY/ABSTRACT Organisms are constantly exposed to environmental conditions that challenge the integrity of the genome. Loss of genomic integrity contributes to the development of most cancers. DNA double strand breaks (DSBs) are a dangerous type of DNA damage that can lead to rapid loss of sequence information stored within the genome. Homologous recombination (HR) is one of the primary DSB repair pathways and is predicated on locating an undamaged DNA sequence that matches the damaged DNA sequence elsewhere in the genome. The homologous sequence can then be used to restore the lost DNA sequence information. During normal mitotic growth, HR preferentially repairs DSBs using sequence information stored in the sister chromatid. Aiding in maintenance of allelic variation between genes and preventing unbalanced exchange of genetic information between chromosomes. In contrast during meiosis the homologous chromosome becomes the preferred DNA repair substrate. There is a large amount information on existing pathways that have evolved in S. cerevisiae to promote DNA repair from the homologous chromosome during meiosis. However, little is known about how homologous chromosomes are used for repair in humans. One of the key determinants in chromosome choice during HR, is the organization of the presynaptic complex (PSC). The regulation, formation, and activity of the human PSC is controlled by >45 proteins. However, a basic functional unit of the PSC consists of RAD51 and associated factors (RAD54L) during mitosis, and RAD51, DMC1 and their associated factors (RAD54L, RAD54B, HOP2-MND1) during meiosis. Understanding how these proteins organize into active complexes during HR is a critical step in understanding how human homologous chromosomes are used for HR. Over the course of our studies we will use biochemical and single molecule approaches to understand the mechanism behind RAD51 and DMC1 self-segregation during meiotic PSC formation. We will understand how DMC1 forms a meiotic homology search complex, and with cooperation of accessory proteins, aligns DNA sequences. We will identify how meiotic homology search complexes overcome chromatin. Finally, we will work to understand how conflicts between the two highly related motor protein RAD54L and RAD54B may promote homologous chromosome use during human mitotic HR. In summary, the primary goal of this research proposal will be to use molecular biology, biochemistry, and single molecule approaches to understand how human mitotic and meiotic PSCs organize, and promote DNA sequence alignment during HR. The data we collect from these experiments will be used to build a model for how human homologous chromosome selection may occur during both mitotic and meiotic HR.