RNA Polymerase Transcription Past DNA Adducts

描述(申请人提供)：这项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修复和转录本完整性的水平上评估随后的细胞反应。当RNA聚合酶在基因组中的损伤处失速时，它们充当DNA损伤的传感器，有时通过转录耦合DNA修复触发损伤清除，这与核苷酸切除修复重叠，需要至少两种在Cockayne综合征中有缺陷的额外蛋白质。但TCR与其他DNA修复途径如碱基切除修复的重叠还没有得到明确的证明或否定。与阻碍转录复合体进展的DNA损伤相反，DNA中的一些损伤允许部分或完全转录旁路，导致产生全长RNA，其中可能包含碱基错误插入或缺失，从而可能通过“转录突变”损害新生转录本的功能。这种对mRNA的改变可能导致蛋白质的改变，从根本上影响细胞生理，可能引发疾病。因此，与表达基因中DNA损伤的转录受损相关的健康相关问题是潜在的严重的，然而我们对人类细胞这一领域的基本理解是相当有限的。在这一应用中，我们建议进行实验，以检测DNA损伤对转录的影响，并进一步破译转录偶联DNA修复的机制。这项工作将在人类细胞中完成，使这项工作超越了迄今使用的生化方法。实现这些目标有三个具体目标。我们将：(1)通过选择DNA加合物来研究RNA聚合酶II的转录；(2)确定通过绕过每个病变产生的mRNA的碱基序列；以及(3)检查DNA修复，包括在定点修饰的载体中的TCR。计算机模拟研究将通过提供RNA聚合酶II遇到DNA加合物时的分子模型，在继续解释我们的结果方面发挥作用。这项研究将长期以来的重点从DNA损伤对DNA复制的影响转移到DNA损伤在RNA合成中的作用，RNA合成是一个发生在所有细胞中的过程，包括那些正在进行分裂或末端分化的细胞。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。虽然这类药物经常与突变和癌症有关，但它们很可能对未分裂的细胞构成威胁，并扰乱生长和发育过程中的RNA合成，增加对人类健康的影响。 与公共健康相关：内源性和外源性化学物质损害DNA，损害其存储信息和在细胞内传递信息的能力。这项研究研究了细胞如何修复这种损伤，保存DNA并允许基因正常运作。这些研究将提高我们对癌症和发育性疾病的理解。

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.