Many human genetic diseases that involve defects in processing DNA damage are associated with severe developmental, neurological and immunological abnormalities as well as frequent cancer predisposition. These facts imply an as-yet poorly understood requirement for DNA repair in normal development in addition in its better documented role in prevention of carcinogenesis. This requirement is proposed to be related to damage to DNA from reactive oxygen species generated during metabolism. Because of qualitative similarity in the lesions induced by ionizing radiation and cellular oxidation, understanding the mechanisms for repair of ionizing radiation damage can contribute to understanding this vital role of repair processes. The broad objective of this research program is to elucidate the molecular mechanisms involved in transcription-coupled repair in mammalian cells of DNA lesions induced by ionizing radiation and other agents that produce oxidative damage. In addition, the relationship of transcription-coupled repair of oxidative damage to other DNA transactions and to normal growth and development will be investigated. The hypotheses to be tested are 1) that transcription-coupled repair of oxidative base damage proceeds by utilizing components of both base excision repair (BER) and nucleotide excision repair (NER) pathways, which have classically been regarded as distinct, and 2) that failure to rapidly repair such damage in critical active genes contributes both to cellular end points and to the profound clinical abnormalities of Cockayne syndrome (CS). It has recently been shown that the xeroderma pigmentosum group G (XPG) gene product is essential for removal of the oxidatively damaged base TG from active DNA and that defects in this repair process correlate with the clinical appearance of CS. It is proposed to continue these studies to determine the role of XPG in the transcription-coupled repair of TGs, to identify other required functions, to assess the generality of the process in repair of other oxidatively damaged bases, and to further explore the relationship between defective repair of oxidative damage and the severe abnormalities of CS. Immediate objectives include 1) investigation of the mechanism of XPG-dependent TG removal to distinguish between a role of XPG in assembly of proteins for base excision repair vs. a direct role in incision; 2) examination of a possible overlap between this process and a PCNA-dependent pathway of base excision repair; and 3) investigation of normal and mutant cellular responses to ionizing radiation to evaluate possible mechanisms by which defective transcription-coupled repair of oxidative lesions in DNA could produce developmental defects.
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