DNA damage recognition by nucleotide excision repair proteins

核苷酸切除修复蛋白识别 DNA 损伤

基本信息

批准号：
8897805
负责人：
Bennett Van Houten
金额：
$ 38.83万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-09 至 2020-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8897805
关键词：
Ablation Aromatic Polycyclic Hydrocarbons Atomic Force Microscopy Back Binding Cell Extracts Cells Cisplatin Cockayne Syndrome Collaborations Complex DNA DNA Binding DNA Damage DNA Repair DNA Repair Gene DNA lesion Diffuse Diffusion Dissociation Eukaryotic Cell Genes Genetic Genetic Materials Hand Human In Vitro Kinetics Label Lesion Life Malignant Neoplasms Measures Mediating Modeling Mutation Nature Nucleotide Excision Repair Oncogenic Pathway interactions Pharmacologic Substance Process Protein Array Proteins Quantum Dots RBX1 gene Reading Repair Complex Research Saccharomyces cerevisiae Saccharomycetales Site Site-Directed Mutagenesis Skin Cancer Stretching Structure System Techniques Testing Time Trichothiodystrophy UV induced Work XPA gene Xeroderma Pigmentosum Yeasts base human DNA human disease innovation microscopic imaging novel protein complex public health relevance repaired single molecule

项目摘要

DESCRIPTION (provided by applicant): This highly innovative project consists of four aims and investigates the dynamics of eukaryotic nucleotide excision repair proteins at the single molecule level. Specifically, this study analyzes DNA damage through five discrete steps involving: i) initial non-target DNA binding by a NER recognition complex; ii) diffusion of this repair complex to a lesion site; iii) lesion processing, "conformational proof reading" by this complex (3) (such as insertion of a beta-hairpin into the DNA (Rad4/XPC); iv) arrival of a DNA repair recognition complex ; and v) lesion processing by the second repair complex causing the first repair complex to diffuse away from the damaged site. We hypothesize that the efficiency of the hand-off from one protein complex to the next is the rate-limiting step(s) in NER. Aim 1 investigates the interactions of purified Rad4-Rad23-Rad33 or XPC- RAD23B-CETN2 complexes with damaged DNA. The second aim measures the kinetic interactions of Rad14 or XPA with damaged DNA. The particularly original third aim studies the interaction of specific NER damage recognition components in whole-cell extracts of Saccharomyces cerevisiae. Genetic ablation or site-directed mutation into specific genetic loci will allow analysis of the important domains that are essential for damage recognition and lesion hand-off. The fourth aim measures the damage handoff from UV-DDB to XPC-RAD23B and XPA and how ubiquitylation or PARylation stimulates this process. This project will give an unprecedented view of the complex process of damage recognition steps of eukaryotic nucleotide excision repair and answer several key questions regarding damage recognition that have been intractable in the absence of single molecule approaches. Completion of this project will have a long and lasting impact on the field.

描述（由适用提供）：这个高度创新的项目由四个目的组成，并研究了单个分子水平上真核核核肽惊喜修复蛋白的动力学。具体而言，本研究通过涉及以下五个离散步骤来分析DNA损伤：i）NER识别复合物的初始非目标DNA结合； ii）将这种修复复合物扩散到病变部位； iii）病变处理，该复合物（3）（例如将β-发hairpin插入DNA（RAD4/XPC）; iv）到达DNA修复识别复合物的到达； v）通过第二修复复合物进行病变处理，导致第一个修复复合物从受损部位扩散。我们假设从一个蛋白质复合物到下一个蛋白质复合物的交接效率是NER中的速率限制步骤。 AIM 1研究了纯化的RAD4-RAD23-RAD33或XPC-RAD23B-CETN2复合物与受损DNA的相互作用。第二个目标测量了RAD14或XPA与受损DNA的动力学相互作用。特别原始的第三目研究了酿酒酵母全细胞提取物中特定NER损伤识别成分的相互作用。遗传消融或位于特定遗传基因座的位置突变将允许分析对损害识别和病变处理至关重要的重要域。第四个目标衡量了从UV-DDB到XPC-RAD23B和XPA的损坏交接，以及泛素化或pary液如何刺激这一过程。该项目将对真核核底尿素惊喜修复的损害识别步骤的复杂过程进行前所未有的看法，并回答有关损害识别的几个关键问题，这些问题在没有单分子方法的情况下是可悲的。该项目的完成将对该领域产生较长而持久的影响。