Much of our effort has been to define the structure-function mechanisms and biochemical properties of Ape1, the major protein for repairing abasic sites and certain 3-damages in DNA. Our work has revealed that Ape1 cleaves at AP sites in single-stranded regions of complex, biologically-relevant DNA structures, such as bubble and fork intermediates. These findings expand the known repertoire of substrates processed by this enzyme, and suggest novel functions for Ape1 likely coupled to transcription and/or replication. We recently found that the protein defective in the human segmental progeroid, Cockayne Syndrome B (CSB), physically and functionally interacts with Ape1. We are now determining the precise molecular contributions of CSB to BER and testing more exhaustively for possible helicase- and remodeling-type activities for CSB on a variety of DNA and RNA substrates. These studies in total will determine the molecular functions of CSB and how certain activities contribute to the associated disease manifestation. Last, we are designing methods to strategically regulate Ape1 repair activity using a dominant-negative approach in the hopes of developing more effective anti-cancer treatment paradigms.? ? In addition to the investigations above, we are elucidating the biochemical and cellular contributions of XRCC1, a major SSB repair (SSBR) factor. This protein functions primarily as a scaffold component, orchestrating specific protein-protein interactions required for efficient DNA repair. Our studies (i) suggest a link of XRCC1 to replication via an interaction with PCNA, (ii) argue against a role for XRCC1 in the early steps of BER, and (iii) indicate a biologically-relevant role for its interaction with DNA polymerase beta and in the subsequent repair step, nick ligation. Recent work has identified associations of XRCC1 with proteins defective in human neurodegenerative disorders AOA1 (Aprataxin) and SCAN1 (TDP1). We are assessing the role of XRCC1 in non-dividing (neuronal) cells and age-related pathologies (namely neurodegeneration) using cell culture models and heterozygous mice. The contributions of XRCC1 to DNA damage responses, chromosome stability, and telomere maintenance are concurrently being evaluated in dividing human cells using chronic RNAi knockdown strategies. The ongoing investigations will delineate the contribution of SSBs (i.e. an XRCC1 deficiency), a common DNA intermediate, to cancer promotion (genetic stability) and neurodegenerative disease (neuronal cell viability).
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