The long-term objective of this project is to better understand what governs the efficiency with which DNA damage is repaired in specific regions of the genome. Although the general pathways for excision repair of various classes of structural defects in mammalian cells have been worked out, it is only the """"""""average"""""""" response of the entire genome that has been assayed in most repair studies. However, it is important to understand in detail how the excision repair responses might be governed by unique features or regions of the genome, such as the status of expression of structural genes. The overall experimental strategy is to simplify the analysis by using defined DNA sequences so that the processing of damage can be analyzed at the molecular level. The basic approach used to study repair in specific sequences involves the physical separation of DNA regions containing bromodeoxyuridine (BrdUrd) substitution in repair patches from all other DNA using a monoclonal antibody against BrdUrd. Specific (32)P-labeled probes are used to detect and quantitate very small amounts of the sequence(s) of interest in each fraction. This technique sensitively detects the repair synthesis event itself and, therefore, allows direct comparison of repair in specific sequences of a variety of types of DNA damage. The research approach described in this proposal is designed to: (1) Determine what role transcription plays in initiating preferential repair of actively transcribed genes by elucidating the mechanism by which an inhibitor of RNA polymerase II eliminates preferential repair on the transcribed strand of a gene and by examining repair of DNA sequences containing the regulatory elements for a gene. (2) Determine whether preferential repair of active genes occurs in lower eukaryotes as it does in mammalian cells. If this is found to be the case, we will develop a model using relevant mutants of the yeast Saccharomyces cerevisiae to directly assess the role of transcription on the efficiency of DNA repair and the biological implications of this repair pathway. (3) Determine the relationship between the metabolism of polycyclic aromatic hydrocarbons and the production of oxidative DNA damage, and whether oxidative DNA damage is preferentially repaired in active genes.
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