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RNA Polymerase Transcription Past DNA Adducts

RNA 聚合酶转录 DNA 加合物

基本信息

批准号：
8002023
负责人：
David A Scicchitano
金额：
$ 29.46万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-09-30 至 2013-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8002023
关键词：
4-nitroimidazole Address Adopted Affect Area Aromatic Polycyclic Hydrocarbons Base Excision Repairs Base Sequence Behavior Biochemical Biological Biometry Bypass Cell physiology Cells Chemicals Cockayne Syndrome Collaborations Complex Computer Simulation Coupled DNA DNA Adducts DNA Damage DNA Repair DNA Repair Pathway DNA Sequence DNA biosynthesis DNA lesion DNA-Directed RNA Polymerase Data Defect Development Diagnostic Disease Elements Elongation Factor Enzymes Epoxy Compounds Event Excision Exhibits Fibroblasts Gene Expression Genes Genetic Transcription Genome Glycols Goals Green Fluorescent Proteins Growth Growth and Development function Health Human Hydrogen Bonding In Vitro Interphase Cell Ionizing radiation Knowledge Length Lesion Letters Light Link Malignant Neoplasms Mathematics Messenger RNA Molecular Conformation Molecular Models Monitor Mus Mutagenesis Mutagens Mutation Nature Neurologic New York Nucleic Acid Regulatory Sequences Nucleotide Excision Repair Pathway interactions Peroxonitrite Plasmids Play Population Premature aging syndrome Process Production Protein Biosynthesis Proteins Publishing RNA RNA Polymerase II RNA chemical synthesis Reaction Regulatory Element Reporter Reporter Genes Research Role Site System Testing Transcript Transcription Elongation Transcription Process Translating Translations Ultraviolet Rays Universities Work adduct base biochemical model chemical property coping experience flexibility gene synthesis human disease improved meetings molecular modeling promoter protein expression protein function public health relevance red fluorescent protein repaired research study response sensor tool vector

项目摘要

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.

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转录过程中 RNA 聚合酶的行为，并评估随后在 DNA 修复和转录完整性水平上的细胞反应。 RNA聚合酶在基因组损伤处停滞时充当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，并且需要至少两种在科凯恩综合征中存在缺陷的额外蛋白质。但 TCR 与其他 DNA 修复途径（例如碱基切除修复）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旁路，导致产生可能包含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 的这种变化可能会导致蛋白质发生改变，从而从根本上影响细胞生理学，甚至可能引发疾病。因此，与表达基因中 DNA 损伤后的转录受损相关的健康相关问题可能很严重，但我们对人类细胞这一领域的基本了解却相当有限。在此应用中，我们提出实验来检查 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。这项工作将在人体细胞中完成，超越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）研究经过选择的 DNA 加合物的 RNA 聚合酶 II 转录； (2)确定各病灶旁路产生的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。计算机建模研究将通过提供 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（这对于细胞的生长和分裂很重要）转移到了DNA损伤在RNA合成中的作用，RNA合成是所有细胞中都发生的过程，包括那些正在分裂或终末分化的细胞。这项研究将加深我们对环境基因毒性剂对人类转录的有害影响的了解。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，并扰乱生长和发育过程中的 RNA 合成，从而增加对人类健康的影响。公共卫生相关性：内源性和外源性化学物质会损害 DNA，损害其在细胞内存储信息和传输信息的能力。这项研究研究细胞如何修复这种损伤，保存 DNA 并让基因正常发挥作用。这些研究将增进我们对癌症和发育疾病的了解。