Living cells have evolved an elaborate array of enzymatic systems to maintain the integrity of their genetic material in the face of numerous environmental agents that alter DNA structure or base sequence. In addition to damage produced by environmental agents such as radiation and reactive chemicals, DNA is subject to deleterious modification by endogenous events occurring naturally in the cellular environment. Although the general pathways for excision repair of various classes of structural defects in eukaryotic cells have been worked out, it is only the """"""""average"""""""" response of the entire genome that has been assayed in most repair studies. The long term objective of this project is to provide a better understanding of the molecular mechanisms involved in the repair of DNA damage in specific DNA sequences. Our hypothesis is that some proteins essential for global genome repair of particular lesions may also have separate functions in transcription-coupled repair that can therefore only be detected by examining the repair of a variety of types of DNA damage. The proposed investigations emerge from our recent findings that DNA damage induced by ionizing radiation is preferentially repaired in active genes. This preferential repair occurs in both human and yeast cells and is due to a faster rate of repair on the transcribed strand of the gene compared to the nontranscribed strand or the genome overall. We find that genes defective in Cockayne syndrome (CS), xeroderma pigmentosum (XP) group G patients that also clinically exhibit CS (XP/CS complex), and human DNA mismatch repair mutants are differentially involved in the transcription-coupled repair of ionizing radiation damage. We propose to continue our studies of transcription-coupled repair using mutants of both human cells and the yeast Saccharomyces cerevisiae, which has proven to be a versatile eukaryotic paradigm for molecular studies of DNA- repair. The proposed research is designed to: (1) Examine the defects in transcription-coupled repair in cells derived from CS and XP/CS individuals, and in yeast which contain mutations in the genes homologous to those defective in CS and XP. Repair will be examined in the transcribed and nontranscribed strands of active genes using immunological approaches that detect either the repair synthesis step or a specific DNA lesion. (2) Determine whether products of DNA mismatch repair genes in human and yeast cells are involved in.transcription-coupled repair. A model suggesting that the recognition of RNA polymerase stalled at a DNA lesion by the mismatch repair system could account for the preferential repair of both ionizing radiation and UV damage will be tested in both human and yeast cells. (3) Investigate transcriptioncoupled repair of DNA lesions that block RNA polymerase with varying efficiencies. Using an immunological approach, the repair of thymine glycols (a strong block to RNA polymerase), 8-oxo-guanine (an intermediate block), and 8-oxo-adenine (a weak block) will be compared in transcriptionally active DNA in both human and yeast cells. Additionally, human and yeast cells in which the AP endonuclease activity is reduced or absent will be examined for their ability to carry out transcription-coupled repair.
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