) DNA synthesis and ligation are required to complete the homologous recombinational repair of DNA double-strand breaks (DSBs). These processes respectively replace the genetic information lost during the nucleolytic processing of DSBs, and restore the integrity of the phosphodiester backbone of the recombining molecules. We hypothesize that DNA synthesis and ligation are temporally coupled in a DNA damage-dependent manner to the initial steps of homologous recombination, in particular heteroduplex formation. We will systematically search for associations between recombination and DNA synthesis proteins and determine whether these associations are modulated in a DNA damage-dependent manner by a combination of in vitro and in vivo approaches. The effects of complex formation between recombination and DNA synthesis proteins on the functional properties of the interacting proteins will be determined. Specifically we will examine whether novel complexes of recombination and DNA synthesis proteins can catalyze heteroduplex formation and strand invasion-dependent synthesis. In collaboration with Project 1 we will reconstitute strand invasion-dependent DNA synthesis with purified recombination proteins and DNA polymerases with their accessory factors. The putative role of DNA ligase I in the cellular response to ionizing radiation and, more specifically, in the completion of homologous recombination repair will be examined in studies with a DNA ligase I-deficient human cell line. The goal of this study is to elucidate the molecular mechanisms by which homology-dependent repair of DSBs is completed. This information will form the basis for understanding how this pathway is regulated in normal cells and how abnormalities in this pathway result in cancer formation. This project will be conducted in close collaboration with Projects 1, 3, and 4, and will require the services of the animal, macromolecular synthesis and analyses, and imaging cores.
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