The Project (Nucleotide Excision Repair and Base Excision Repair) focuses on understanding the interactions of key proteins with DNA substrates and partner proteins critical for functional DNA excision repair complexes. Our goal is to leverage structural and biochemical studies of the repair endonuclease ERCC1-XPF and of DNA ligase to illuminate the coordination of the multi-step reaction pathways that comprise Nucleotide Excision Repair (NER) and the completion of B_ase Excision Repair (NER). Defects in DNA excision repair proteins and pathways result in increased rates of mutation, chromosomal breakage, and an increased incidence of cancers. Distortions of the helical structure of DNA are specifically recognized by repair enzymes and can be read out in a way that does not depend on the chemical identity of the damage. This generalized strategy enables one enzyme to initiate the repair of a variety of lesions in DNA. Moreover, interactions with other DNA binding and repair proteins provide additional biological specificity and contribute to the efficiency of repair. Although the enzymatic activities constituting the basic NER and BER pathways are known, it remains to be determined how these activities are coordinated into a multi-step reaction pathway by the physical interactions within enzyme-DNA complexes catalyzing the excision repair of DNA damage. Structural analyses of the relevant enzyme-DNA complexes will reveal distinct conformational states of the enzymes and their DNA substrates corresponding to different steps of the repair reaction. Low-resolution structures and conformations derived from x-ray scattering in solution will complement high-resolution images of the repair complexes that can be crystallized. In this way, the dynamic assembly and disassembly of multi-protein complexes catalyzing DNA repair will be characterized. We propose to test and develop these hypotheses by investigating specific excision repair components as follows: 1) Catalytic Substrate specificity of XPF-ERCC1, and related DNA structure-specific nucleases;2) Damage senses by XPA that recruits XPFERCC1 to NER complexes;3) Catalytic selectivity of DNA ligases;Nick-sensing and DNA repair;4) Interactions of DNA Ligase I with DNA sliding clamps;and 5) Interactions of ligase I with the clamp loaders.
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