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中共价修饰的碱基如何影响RNA聚合酶在转录过程中的行为，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聚合酶在基因组中的病变处停滞时，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，并且需要至少两种在疾病Cockayne综合征中有缺陷的额外蛋白质。但是TCR与其他DNA修复途径如碱基切除修复的重叠还没有被明确证明或反驳。与阻止转录复合物进展的DNA损伤相反，DNA中的一些损伤允许部分或完全转录旁路，导致产生可能含有碱基错误插入或缺失的全长RNA，可能通过“转录诱变”损害新生转录物的功能。“mRNA的这种变化可能导致蛋白质的改变，从而从根本上影响细胞生理学，可能引发疾病。因此，与表达基因中的DNA损伤后的转录受损相关的健康相关问题可能是严重的，但我们对人类细胞中这一领域的基本了解非常有限。在本申请中，我们提出了实验来检查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，超越了迄今为止使用的生物化学方法。实现这些目标有三个具体目标。我们将：（1）研究RNA聚合酶II转录经过选择的DNA加合物;（2）确定通过绕过每个病变产生的mRNA的碱基序列;（3）检查位点特异性修饰载体中的DNA修复，包括TCR。计算机模拟研究将发挥作用，在继续解释我们的结果，提供分子模型的RNA聚合酶II时，遇到的DNA加合物。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，包括那些正在经历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因子对人类转录的有害影响的理解。虽然这些物质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，从而增加其对人类健康的影响。

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.