HUMAN RNA POLYMERASE II TRANSCRIPTION PAST PAH ADDUCTS

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In this new application, Dr. Scicchitano proposes to elucidate mechanisms by which covalently modified bases in DNA cause human RNA polymerase II to stall at the altered site, or allow it to progress past the lesion, resulting in the formation of full-length transcripts. It is surmised that stalling of RNA polymerase II at polycyclic aromatic hydrocarbon (PAH) adducts present in transcribed DNA is not merely a function of presence of the lesion in the template, but is related to the three-dimensional orientation of the adduct and the base incorporated into the transcript at the damaged site. The following aims are proposed to address these ideas: 1) Templates suitable for transcription by RNA polymerase II present in nuclear extracts will be prepared. They will contain the CMV immediate early promoter/enhancer element, a G-less cassette, and a site-specific stereochemically pure PAH adduct. 2) The templates will be used in transcription run-off assays to assess potential blocks to RNA synthesis and relative amounts of bypass. 3) The base sequence of full-length transcripts obtained by elongation past the modified base in DNA will be determined; this will test for nucleotide incorporation at or near the adduct. 4) The base sequence at the 3' ends of truncated transcripts will be characterized. Truncated transcripts often form as a result of RNA polymerase stalling due to the presence of a lesion. Finally, (5) transcription past site-specific PAH lesions will be studied using extracts from cells derived from patients with Cockayne's syndrome, a disease characterized by the inability to carry out rapid repair of DNA adducts present in expressed genes. The data will be interpreted in light of information concerning the mutagenicity, tumorigenicity, repair, and structural features of the DNA adducts being studied. They will also help to gain a better understanding of the role of transcription as a biological endpoint adversely affected by these highly carcinogenic agents.