HUMAN RNA POLYMERASE II TRANSCRIPTION PAST PAH ADDUCTS

在这项新的应用中，Scicchitano博士提出要阐明 DNA中共价修饰的碱基导致人类RNA聚合酶 II在改变的部位失速，或允许它通过病变， 导致全长转录物的形成。据推测， RNA聚合酶II在多环芳烃（PAH）加合物上的停滞 在转录的DNA中的存在不仅仅是损伤存在的功能 在模板中，但是与模板的三维取向有关。 加合物和碱在受损位点掺入转录物中。的 提出了以下目标来解决这些想法：1）模板适合 核提取物中RNA聚合酶II的转录将是 制备它们将含有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测定，以评估潜在的 阻断RNA合成和旁路的相对量。3)的碱基序列 通过延伸超过DNA中修饰的碱基而获得的全长转录物 将被确定;这将测试核苷酸掺入或附近的 加合物4)截短的转录物的3'端的碱基序列将是 表征了截短的转录物通常是RNA聚合酶的结果 由于病变的存在而停止。最后，（5）转录过去 将使用来自细胞的提取物研究部位特异性PAH病变， 来自Cockayne综合征患者，这种疾病的特征是 不能对表达基因中存在的DNA加合物进行快速修复。 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 研究加合物。它们也将有助于更好地了解 转录作为生物学终点的作用受到这些不利影响， 高度致癌物质。

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.