HUMAN RNA POLYMERASE II TRANSCRIPTION PAST PAH ADDUCTS

人类 RNA 聚合酶 II 转录 PAH 加合物

• 批准号: 6382376
• 负责人: David A Scicchitano
• 金额: $ 25.79万
• 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/6382376
• 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.

在这项新应用中，Scicchitano 博士建议阐明机制 DNA 中共价修饰的碱基引起人类 RNA 聚合酶 II 在改变的部位停止，或让它前进越过病变， 从而形成全长转录本。据推测 RNA 聚合酶 II 在多环芳烃 (PAH) 加合物处失速 存在于转录 DNA 中的 DNA 不仅仅是病变存在的函数 在模板中，但与三维方向有关 加合物和碱基并入受损位点的转录物中。这 提出以下目标来解决这些想法：1）适合的模板 核提取物中存在的 RNA 聚合酶 II 的转录将是 准备好了。它们将包含 CMV 立即早期启动子/增强子元件， 无 G 盒和位点特异性立体化学纯 PAH 加合物。 2) 的 模板将用于转录径流分析以评估潜力 阻止 RNA 合成和相对数量的旁路。 3) 碱基序列 通过延伸超过 DNA 中修饰碱基获得的全长转录本 将被确定；这将测试在或接近的核苷酸掺入 加合物。 4) 截短转录物 3' 端的碱基序列将是 特点。 RNA 聚合酶通常会形成截短的转录本 由于病变的存在而停滞。最后，（5）转录过去 将使用源自细胞的提取物来研究位点特异性 PAH 病变 来自科凯恩综合征患者，这种疾病的特征是 无法快速修复表达基因中存在的 DNA 加合物。 数据将根据有关信息进行解释 DNA 的致突变性、致瘤性、修复和结构特征 正在研究的加合物。他们也将有助于更好地了解 转录作为生物终点的作用受到这些因素的不利影响 高度致癌剂。