Using basic molecular and biochemical approaches, I and my colleagues have contributed to defining how specific human, core BER proteins recognize and process target lesions and/or coordinate with other components of the pathway. This research has centered largely on apurinic/apyrimidinic endonuclease 1 (APE1), the major mammalian protein for repairing abasic sites in DNA, and x-ray cross-complementing 1 (XRCC1), a key non-enzymatic scaffold protein that facilitates the efficient execution of SSB repair. In addition, we have increased our effort to delineate the role of Cockayne syndrome B (CSB) protein in the processing of endogenous DNA damage. Our studies in recent years have (i) uncovered novel biochemical properties associated with the APE1 repair protein, including its ability to incise at AP sites in certain biologically-relevant DNA configurations; (ii) identified APE1 as a potential target for the genotoxic and co-carcinogenic effects of lead, an important environmental toxin; (iii) established a novel dominant-negative form of APE1 that has potential utility in gene-therapy treatment paradigms; (iv) described the biological significance of specific interactions (e.g. with POLbeta) of the major SSBR protein, XRCC1; (v) reported an interaction of XRCC1 with the replication/repair protein, PCNA, establishing a novel link between the DNA repair machinery and replication factories; (vi) determined the major biochemical repair defect(s) associated with XRCC1 deficiency in mammalian cells; (vii) characterized the repair capacity and genetic stability of human cells deficient for XRCC1 function using transient RNAi knockdown strategies; and (viii) demonstrated that CSB has a physical and functional interaction with APE1. The present objectives of the laboratory as related to this project are described next.? ? APE1 is the major mammalian enzyme responsible for the repair of abasic sites in DNA, and has functions in SSB repair and other cellular processes, including transcriptional regulation. We are in the process of establishing stable shRNA knockdown cell lines to dissect out the precise contribution of each proposed function of APE1 (i.e. its nuclease activity, redox regulatory role, etc.) in cell growth/viability and protection against DNA-damaging agents. In addition, using a dominant-negative form of the APE1 protein (termed ED) and identified small molecular inhibitors, our laboratory will evaluate the contribution of the BER pathway to clinical DNA-damaging agent resistance. My group is also assessing the potential relationship of reduced BER capacity to disease development using established and in-development repair assays and defined population groups. The overall hypothesis is that APE1, and BER more generally, plays a critical role in dictating cellular responsiveness to genotoxic insults and susceptibility to disease manifestation, particularly in the face of certain environmental exposure(s).? ? As for XRCC1, my laboratory is currently assessing the role of this protein in age-related pathologies using heterozygous mice, and will also evaluate the effect of XRCC1 haploinsufficiency on neurodegeneration and cancer proneness following defined insults. This area of investigation has gained heightened interest in lieu of the recent discovery that XRCC1 associates with proteins (TDP1 and Aprataxin) defective in inherited spinocerebellar ataxias. The contributions of XRCC1 to DNA damage responses, genomic stability and telomere maintenance are also being evaluated in defined human cell lines using chronic shRNA knockdown strategies. Our hypothesis is that defects in SSBR will be more deleterious to non-dividing, terminally-differentiated cells than dividing cells, because dividing cells repair SSBs not only by SSBR, but also by replication-mediated HR.? ? Finally, CS is a rare, autosomal recessive disorder characterized by growth failure, impaired development of the nervous system, cutaneous photosensitivity and premature aging. The CSB protein harbors seven helicase-like ATPase motifs found within the SWI2/SNF2 superfamily of chromatin remodeling proteins, and interacts with a number of BER protein factors, yet its precise biochemical and cellular functions remain unclear. My group, in collaboration with Dr. Bohr, recently identified a novel interaction between CSB and APE1. We are currently examining the in vitro activities of CSB on key DNA and RNA transaction intermediates, and will elucidate the contributions of the unique N- and C-terminal portions of the protein that likely impart functional specificity. The hypothesis is that CSB operates as an auxiliary protein in BER by inducing topological changes in nucleic acid targets and directing specific protein interactions, and that these functions are critical to the manifestation of the inherent disease pathologies associated with CS patients.
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