RNA Polymerase Transcription Past DNA Adducts

描述（由申请人提供）：本研究的长期目标是详细了解DNA中共价修饰的碱基在转录过程中如何影响RNA聚合酶的行为，并在DNA修复和转录完整性水平上评估随后的细胞反应。当RNA聚合酶在基因组的损伤处停止时，它们充当DNA损伤的传感器，有时通过转录偶联DNA修复触发损伤清除，这与核苷酸切除修复重叠，需要至少两种额外的蛋白质，这些蛋白质在柯凯因综合征中是有缺陷的。但TCR与其他DNA修复途径（如碱基切除修复）的重叠尚未得到明确证明或反驳。与阻碍转录复合物进展的DNA损伤相反，DNA中的一些损伤允许部分或完全转录绕道，导致全长RNA的产生，可能包含碱基错误插入或缺失，潜在地通过“转录突变”损害新生转录物的功能。mRNA的这种变化会导致蛋白质的改变，从而从根本上影响细胞生理，可能引发疾病。因此，与转录受损相关的健康问题在表达基因中过去的DNA损伤是潜在的严重问题，但我们对人类细胞这一领域的基本了解相当有限。在这个应用程序中，我们提出了实验来研究DNA损伤对转录的影响，并进一步破译转录耦合DNA修复的机制。这项工作将在人类细胞中进行，使这项工作超越了迄今为止使用的生化方法。实现这些目标有三个具体目标。我们将：(1)通过选择DNA加合物研究RNA聚合酶II的转录；(2)确定各病变绕道产生的mRNA的碱基序列；(3)检测位点特异性修饰载体中的DNA修复，包括TCR。通过提供RNA聚合酶II遇到DNA加合物时的分子模型，计算机建模研究将在继续解释我们的结果中发挥作用。这项研究将长期以来的重点从DNA损伤对DNA复制的影响（这在细胞生长和分裂中很重要）转移到DNA损伤在RNA合成中所起的作用，这一过程发生在所有细胞中，包括正在分裂或终末分化的细胞。这项研究将增加我们对环境遗传毒性物质对人类转录的有害影响的理解。虽然这类制剂通常与突变和癌症有关，但它们很可能对非分裂细胞构成威胁，并在生长和发育过程中干扰RNA合成，增加了它们对人类健康的影响。

DESCRIPTION (provided by applicant): The long range goal of this research is to gain a detailed understanding of how covalently modified bases in DNA affect RNA polymerase behavior during transcription, and to assess the subsequent cellular responses at the level of DNA repair and transcript integrity. RNA polymerases act as sensors of DNA damage when they stall at lesions in the genome, sometimes triggering damage clearance via transcription-coupled DNA repair, which overlaps with nucleotide excision repair and requires at least two additional proteins that are defective in the disease Cockayne syndrome. But the overlap of TCR with other DNA repair pathways such as base excision repair has not been unequivocally demonstrated or disproved. In contrast to DNA damage that stalls transcription complex progression, some lesions in DNA permit partial or complete transcriptional bypass, resulting in the production of full-length RNA that can contain base misinsertions or deletions, potentially compromising the nascent transcript's function via "transcriptional mutagenesis." Such changes to mRNA can result in altered proteins that affect cell physiology in fundamental ways, possibly triggering disease. Hence, the health-related problems associated with compromised transcription past DNA damage in expressed genes are potentially severe, and yet our basic understanding in this area in human cells is quite limited. In this application we propose experiments to examine the effect of DNA damage on transcription and to decipher further the mechanism of transcription coupled DNA repair. This work will be done in human cells, taking the work beyond the biochemical approaches used thus far. There are three specific aims to address these goals. We will: (1) investigate RNA polymerase II transcription past select DNA adducts; (2) determine the base sequence of the mRNA produced via bypass of each lesion; and (3) examine DNA repair, including TCR, in the site-specifically modified vector. Computer-modeling studies will play role in the continued interpretation of our results by providing molecular models of RNA polymerase II when it encounters a DNA adduct. This research shifts the long-standing emphasis from the effects of DNA lesions on DNA replication, which is important in cells undergoing growth and division, to the role DNA damage plays in RNA synthesis, a process that occurs in all cells, including those that are undergoing division or are terminally differentiated. This research will increase our understanding of the deleterious effect that environmental genotoxic agents have on transcription in humans. While such agents are often associated with mutations and cancer, they may well pose threats to non-dividing cells and disturb RNA synthesis during growth and development, adding to their impact on human health. PUBLIC HEALTH RELEVANCE: Endogenous and exogenous chemicals damage DNA, compromising its ability to store information and transmit it within cells. This research studies how cells repair this damage, preserving DNA and permitting genes to function properly. The studies will improve our understanding of cancer and developmental diseases.