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）：这项研究的远距离目标是详细了解DNA中共价修改的基础如何影响转录过程中的RNA聚合酶行为，并评估在DNA修复和转录本完整性水平上的后续细胞响应。当RNA聚合酶停滞在基因组的病变时，它们是DNA损伤的传感器，有时通过转录偶联的DNA修复触发损伤清除，这与核苷酸切除修复重叠，并且需要至少两种其他在疾病Cockayne综合征中有缺陷的蛋白质。但是TCR与其他DNA修复途径（例如碱基切除修复）的重叠尚未明确证明或反驳。与DNA损伤失速的DNA损伤相反，DNA中的某些病变允许部分或完整的转录旁路，导致产生全长RNA，可能包含碱基插入或缺失或缺失，从而可能损害新生的转录本函数，通过“转录杂种”的功能。对mRNA的这种变化会导致以基本方式影响细胞生理的蛋白质改变，可能触发疾病。因此，在表达基因中，与转录损伤的转录相关的与健康相关的问题可能很严重，但是我们在人类细胞中对该领域的基本理解非常有限。在此应用中，我们提出了实验，以检查DNA损伤对转录的影响，并进一步破译转录耦合DNA修复的机理。这项工作将在人类细胞中完成，将工作超出到迄今为止使用的生化方法之外。解决这些目标有三个具体目标。我们将：（1）研究RNA聚合酶II转录，过去选择的DNA加合物；（2）确定通过每个病变旁路产生的mRNA的碱基序列；（3）在特定于定义的修饰载体中检查包括TCR在内的DNA修复。计算机模型研究将通过在遇到DNA加合物时提供RNA聚合酶II的分子模型来对我们的结果的持续解释发挥作用。这项研究将长期的重点从DNA病变对DNA复制的影响转变为在经历生长和分裂的细胞中重要的DNA复制，转变为DNA损伤在RNA合成中的作用，这一过程发生在所有细胞中，包括正在经历分裂或最终区分的细胞。这项研究将增加我们对环境遗传毒性对人类转录的有害影响的理解。尽管这种药物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的合成，从而增加了对人类健康的影响。公共卫生相关性：内源性和外源性化学物质损害DNA，损害了其存储信息并将其传输到细胞中的能力。该研究研究细胞如何修复这种损伤，保留DNA并允许基因正常运行。这些研究将提高我们对癌症和发育疾病的理解。