HUMAN RNA POLYMERASE II TRANSCRIPTION PAST PAH ADDUCTS

在这一新的应用中，西奇塔诺博士建议阐明机制 DNA中的共价修饰碱基通过什么引起人RNA聚合酶 在改变的部位停滞不前，或允许它通过病变， 从而形成全长转录本。据推测， RNA聚合酶II在多环芳烃(PAH)加合物上的停滞 在转录的DNA中的存在不仅仅是病变的存在的函数 在模板中，但与 加合物和碱基合并到受损部位的成绩单中。这个 提出了以下目标来解决这些想法：1)适用于 存在于核提取液中的RNA聚合酶II的转录 准备好了。它们将包含CMV立即早期启动子/增强子元件， 一个无G的盒，和一个位置特定的立体化学纯多环芳烃加合物。2) 模板将用于转录径流分析，以评估潜力 阻断RNA合成和旁路相对量。3)碱基序列 通过延长DNA中修饰的碱基而获得的全长转录本 将被确定；这将测试在或接近 相加。4)截短转录本3‘端的碱基序列为 特色化的。截短的转录本通常是由于RNA聚合酶而形成的 由于损伤的存在而停滞不前。最后，(5)抄写过去 将使用从细胞中提取的提取物来研究特定部位的PAH损伤 来自Cockayne综合征的患者，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。 数据将根据以下信息进行解释： DNA的致突变性、致瘤性、修复和结构特征 正在研究的加合物。它们还将有助于更好地了解 转录作为生物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.