The human genome is subject to constant attack by endogenous and environmental DNA damaging agents. If unrepaired, DNA lesions will give rise to mutations that in turn may lead to cancer formation. A complex network of DNA repair pathways operates to remove DNA lesions. In order to accurately assess the biological significance of exposure to environmental DNA damaging agents, it is necessary to understand the molecular details of the complex cellular response to DNA damage. Although prokaryotes and lower eukaryotes have proven to be useful model systems for higher organisms, there are aspects of the DNA response, including DNA repair pathways that appear to be restricted to higher eukaryotes. For example, lower eukaryotes lack homologs of the genes encoding DNA ligase III, XRCC1, poly (ADPribose) polymerase-1 (PARP-1) and DNA polymerase beta. All of these proteins have been implicated in DNA base excision repair and the repair of DNA single-strand breaks. The goal of this grant is to elucidate the roles of the LIG3 and XRCC1 gene products in maintaining genome stability in mammalian cells. In preliminary studies, PARP-1 and the hRAD50/Mre11/Nbs complex have been identified as partners of DNA ligase III-alpha in somatic cells. In addition, DNA ligase III-alpha has been shown to be phosphorylated in a cell-cycle dependent manner and dephosphorylated in response to DNA damage. On the basis of these results, 4 aims are proposed: (i) to delineate the functional and biological consequences of the interaction between DNA ligase III-alpha and PARP-1; (ii) to elucidate the functional and biological consequences of the interaction between DNA ligase III-alpha and the MRE11 complex; (iii) to characterize cell cycle-regulated and DNA damage-dependent modifications of DNA ligase III-alpha; (iv) to determine the cellular functions of the LIG3 gene products by generating lig3 mutant cell lines and animals. These studies will provide novel insights into DNA transactions that are unique to more complex organisms and will contribute to an overall picture of how DNA repair mechanisms protect against DNA damage and mutations induced by exposure to genotoxic environmental agents.
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