The long range goal of this research is to gain a detailed understanding of how covalently modified bases in DNA affect RNA polymerase behavior during the elongation phase of transcription, with the ultimate objective of producing detailed structural models that portray RNA polymerase interactions at bulky and small adducts found in DNA. The following four Specific Aims will be targeted toward this: (1) to assess the effects of transcription past site-specific lesions on elongation past the damaged or modified site; (2) to determine the base sequence of full-length transcripts, and to characterize the base composition at the 3'- ends of truncated transcripts; (3) to determine Km, and Vrnax values for base addition at DNA adducts during transcription; and (4) to employ computer-modeling techniques to provide characteristics of transcription complexes stalled at DNA adducts. It is now quite clear that certain DNA lesions can cause RNA polymerase to stall at the modified site, resulting in a truncated transcript, or progress past the altered base, producing full-length RNA. Stalled transcription complexes signal transcription-coupled DNA repair. The importance of transcription-coupled DNA repair is evidenced by the clinical phenotype observed when it is aberrant, as is seen in patients with Cockayne's syndrome, a disease characterized by severe growth and developmental defects. There are broad and significant implications for preferential clearance of DNA damage from discrete, active genetic loci: Biases in mutagenesis can exist; correlations of DNA damage and repair with tumorigenesis might be stronger when preferential clearance rates for a particular adduct are used for making the comparisons, rather than total genomic repair; and the actual removal of adducts from the transcribed strand of an expressed gene might be very dependent on their ability to impede RNA synthesis. ? ?
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