HUMAN RNA POLYMERASE II TRANSCRIPTION PAST PAH ADDUCTS

人类 RNA 聚合酶 II 转录 PAH 加合物

• 批准号: 6167227
• 负责人: David A Scicchitano
• 金额: $ 25.49万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-30 至 2003-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6167227
• 关键词: DNA damage; DNA directed RNA polymerase; DNA repair; RNA binding protein; RNA biosynthesis; adduct; carbopolycyclic compound; cytomegalovirus; enzyme activity; genetic enhancer element; genetic promoter element; genetic transcription; human tissue; nuclear runoff assay; nucleic acid sequence; oligonucleotides; phosphoester ligase; polymerase chain reaction; stereochemistry; tissue /cell culture; transcription factor

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.

在这个新的应用中，Scicchitano博士建议阐明机制