DNA damage recognition by nucleotide excision repair proteins

 描述(申请人提供)：这个高度创新的项目由四个目标组成，在单分子水平上研究真核核苷酸切除修复蛋白的动力学。具体地说，本研究通过五个不同的步骤分析DNA损伤，涉及：i)NER识别复合体与非靶标DNA的初始结合；ii)该修复复合体扩散到损伤部位；iii)损伤处理，该复合体的“构象校对”(3)(例如将β-发夹插入DNA(Rad4/XPC))；iv)DNA修复识别复合体的到达；以及v)第二修复复合体的损伤处理导致第一修复复合体扩散离开受损部位。我们假设从一个蛋白质复合体到下一个蛋白质复合体的切换效率是NER中的限速步骤(S)。目的1研究纯化的RAD4-RAD23-RAD33或XPC-RAD23B-CETN2与DNA损伤的相互作用。第二个目的是测量Rad14或XPA与损伤DNA的动力学相互作用。最初的第三个目的是研究酿酒酵母全细胞提取物中特定的NER损伤识别成分之间的相互作用。对特定遗传位点的基因消融或定点突变将使我们能够分析对损伤识别和损伤转移至关重要的重要区域。第四个目的是测量从UV-DDB到XPC-RAD23B和XPA的损伤传递以及泛素化或PAR化如何刺激这一过程。这个项目将对真核核苷酸切除修复的损伤识别步骤的复杂过程进行前所未有的观察，并回答在缺乏单分子方法的情况下一直难以解决的几个关于损伤识别的关键问题。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.