DNA repair-defective genetic diseases result in marked cancer predisposition, and many also produce developmental, neurological, or immunological abnormalities, and/or premature aging. The implied requirements for DNA repair in avoidance of carcinogenesis and in normal development are proposed to be related to DNA damage from reactive oxygen species generated by the cellular metabolism as well as by environmental agents including ionizing radiation. The broad objective of this project is to elucidate both the molecular mechanisms for transcription-coupled repair (TCR) and the processing of oxidative DNA damage in human cells, in order to better understand the human health effects of genetic defects in these processes. The approach involves comprehensive characterization of the multiple critical functions of the highly pleiotropic human DNA repair protein XPG, which plays an integrating role in multiple DNA repair processes. The enzymatic activity of XPG is strictly required in nucleotide excision repair (NER) for making the first incision in removal of UV and bulky carcinogen damage. Defects in this function result in the highly cancer-prone disease xeroderma pigmentosum (XP). XPG also has important non-enzymatic roles in base excision repair (BER) of oxidative DNA damage through coordination and stimulation of early steps in lesion removal and in transcription-coupled repair (TCR) - the preferential repair of lesions of transcribed strands of active genes - through interaction with stalled RNA polymerase and other TCR proteins. Mutations in XPG that inactivate these non-enzymatic functions result in the profound developmental and neurological disorder Cockayne syndrome (CS). The hypotheses to be tested are that (a) XPG together with CSB has a critical role in the early steps of TCR that is important both for responses to environmental DNA damage and for maintenance of genome function under normal growth conditions, and that (b) XPG additionally has biologically important roles in global BER of oxidative damage. Both functions are hypothesized to be important in prevention of radiation- and chemical-induced carcinogenesis involving oxidative DNA damage as well as for normal postnatal development. It is proposed (1) to characterize the roles of XPG and CSB in repair of oxidative DNA damage and determine whether global or transcription-coupled mechanisms, or both, are involved; (2) to investigate the assembly of TCR complexes in cells in response to oxidative DNA damage; (3) to investigate the effect of a CS-related mutant fragment of the TCR protein CSB and its interaction with XPG on cellular responses to oxidative DNA damage and the CS phenotype; and (4) to define early steps in TCR and investigate a proposed remodeling of stalled RNA polymerase II by TCR proteins to enable repair. These studies address key mechanisms for maintaining genomic integrity and function in the face of cellular and environmental oxidative DNA damage. ? ? ?
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