DNA damage recognition by nucleotide excision repair proteins

 描述（由申请人提供）：这个高度创新的项目包括四个目标，并在单分子水平上研究真核生物核苷酸切除修复蛋白的动力学。具体来说，这项研究通过五个离散步骤分析DNA损伤，包括：i）NER识别复合物与初始非靶DNA结合; ii）该修复复合物扩散到损伤部位; iii）损伤处理，该复合物的“构象证明阅读”（3）（如将β-发夹插入DNA（Rad 4/XPC）; iv）DNA修复识别复合物的到达;和v）通过第二修复复合物处理损伤，使第一修复复合物扩散离开受损部位。我们假设从一个蛋白质复合物到下一个蛋白质复合物的传递效率是NER中的限速步骤。目的1研究纯化的Rad 4-Rad 23-Rad 33或XPC-Rad 23 B-CETN 2复合物与损伤DNA的相互作用。第二个目的是测量Rad 14或XPA与受损DNA的动力学相互作用。特别是原始的第三个目的研究特定的NER损伤识别组件的酿酒酵母的全细胞提取物的相互作用。基因消融或定点突变到特定的遗传基因座将允许分析的重要领域，是必不可少的损害识别和病变的手。第四个目标测量从UV-DDB到XPC-RAD 23 B和XPA的损伤传递以及泛素化或PAR化如何刺激这一过程。该项目将提供一个前所未有的视图的复杂过程中的损伤识别步骤的真核核苷酸切除修复，并回答几个关键问题的损伤识别，一直难以在单分子方法的情况下。该项目的完成将对实地产生长期和持久的影响。

 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.