The human genome is subject to constant attack by endogenous and environmental DNA damaging agents. Ifunrepaired, DNA lesions will give rise to mutations that in turn may lead to cancer formation. Fortunately, acomplex network of DNA repair pathways operates to remove DNA lesions. To assess the biologicalsignificance of exposure to environmental DNA damaging agents, it is necessary to understand the details ofthe complex cellular response to DNA damage. Unlike the conserved LIG1 and LIG4 genes, lower eukaryoteslack a homolog of the mammalian LIG3 gene, which encodes at least three distinct polypeptides. Interestingly,the DNA ligase III -associated proteins, poly (ADP-ribose) polymerase 1 (PARP-1), XRCC1 and DNApolymerase (Pol) , each of which have been implicated in base excision repair and the repair of DNA singlestrand breaks, are also found only in higher eukaryotes. Using a modified yeast two hybrid assay, we haveidentified a series of XRCC1 mutants that are defective in specific protein-protein interactions.
In Specific Aim1, we will utilize these mutants to delineate the functional and biological consequences of protein-proteininteractions between DNA ligase III /XRCC1 and other proteins involved in base excision and single strandbreak repair. Recent studies have increased the repertoire of DNA repair transactions in which DNA ligaseIII /XRCC1 participates.
In Specific Aim 2, we will determine how DNA ligase III /XRCC1 is recruited to theDNA nucleotide excision repair machinery and whether this involves an interaction between XRCC1 andPCNA. In preliminary studies, we have identified an interaction between DNA ligase III /XRCC1 andhRad50/hMre11/Nbs.
In Specific Aim 3, we will determine how DNA damage regulates this interaction andwhether these proteins act together in an error-prone non-homologous end-joining sub pathway that repairsDNA double strand breaks. Interestingly, this error-prone pathway is up-regulated in cancer cells and maycontribute to their characteristic genomic instability.
In Specific Aim 4, we will identify and characterize smallmolecule inhibitors of DNA ligase III. We envision that that these inhibitors will not only be valuable reagentsfor elucidating the cellular functions of the LIG3 gene products but also may serve as lead compounds for thedevelopment of novel anti-cancer agents.
It is well established that genomic instability drives the progression from a normal cell into a cancer cell. Human cells have a complex network of pathways that act together to maintain genome stability. A mechanistic understanding of these pathways will provide fundamental insights into tumor suppression. In addition, genomic instability is a characteristic of tumor cells, indicating that there are differences in the pathways that normally maintain stability. These differences between normal and cancer cells offer an opportunity to develop therapeutic strategies that selectively target cancer cells.
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