Mechanism of DNA Damage Recognition in Higher Eukaryotes

顺铂是一种广泛应用的抗癌化疗药物，它通过在细胞内形成顺铂-DNA外展物诱导DNA损伤。体内和体外研究强烈表明，大多数顺铂- dna外展物是通过核苷酸切除修复（NER）途径修复的。经过广泛的努力，我们现在对NER的机制和所涉及的蛋白质有了重要的了解。识别DNA损伤是修复早期的关键步骤。着色性干皮病A族互补蛋白（XPA）、复制蛋白A （RPA）、XPC-hHR23B和XPE可以独立结合受损DNA。然而，受损的识别蛋白如何在受损的DNA位点起作用仍存在争议。在本提案中，我们将使用生化和分子方法来解决以下具体问题：1)多种损伤识别因子如何在受损DNA位点起作用？2)锌指蛋白（RPA和XPA）是否会导致受损部位的结构扭曲？如果是这样，是否需要双切口？3)损伤识别因素如何影响3′和5′切口的效率和准确性？在第一个目标中，将使用纯化的修复蛋白（RPA，XPA，XPC-hHR23B和TFIIH）和顺铂诱导的链内交联DNA来分析单个损伤识别蛋白与受损DNA的结合动力学，受损识别因子之间的相互作用以及切口前复合物的组装。在第二个目标中，我们将分析顺铂损伤DNA结构论文的分子基础。我们将分析损伤识别蛋白与惊奇DNA结合后的构象电荷，以及RPA和XPA的锌指基序在这一事件中的作用。我们将使用足印试验来分析由损伤识别因素引起的受损DNA的结构扭曲。此外，将利用荧光共振能量转移（FRET）方法同时监测损伤识别蛋白的构象变化和损伤DNA的畸变。在第三个目标中，我们将尝试从功能上定义XPG在3‘和5’切口活动中的损伤识别因素的作用，并将检查ERCCI-XPF。3‘和5’切口的精度和效率将通过切口活动动力学提前分析。损伤识别因子（RPA和XPC）的各种突变体将用于检查这些蛋白在3‘和5’切口中所具有的任何独特作用。

Cisplatin is widely used anti-cancer chemotherapeutic drug that induces DNA damage by forming Cisplatin-DNA abducts in cells. In vivo and in citron studies strongly suggest that most of the Cisplatin-DNA abducts are repaired through nucleotide excision repair (NER) pathway. Due to extensive efforts, we now have significant knowledge about the mechanism of NER and the proteins involved. Recognition of DNA damage is a critical step in the early stage of repair. Xeroderma pigmentosum group A complementing protein (XPA), replication protein A (RPA), XPC-hHR23B, and XPE can independently bind damaged DNA. However, it is still in debate how the damaged recognition proteins function at the damaged DNA site. In this proposal, we will use biochemical and molecular approaches to address the following specific questions: 1) how multiple damage recognition factors function at the damaged DNA site? 2) do zinc-finger proteins (RPA and XPA) cause structural distortion at the damaged site? If so, is it necessary for dual incisions? 3) how do the damaged recognition factors affect the efficiency and accuracy of 3' and 5'- incisions? In the first aim, binding kinetics of individual damage recognition proteins to damaged DNA, interactions between damaged recognition factors, and assembly of a preincision complex will be analyzed using purified repair proteins (RPA,XPA,XPC-hHR23B, and TFIIH) and Cisplatin-induced intra strand crossed-linked DNA. In the second aim, we will analyze the molecular basis for structural dissertation of Cisplatin-damaged DNA. Conformational charges of damage recognition proteins following their interment with amazed DNA and the role of the zinc-finger motif of RPA and XPA in this event will be analyzed. We will use a foot printing assay to analyze the structural distortion of damaged DNA induced by damage recognition factors. In addition, fluorescence resonance energy transfer (FRET) method will be utilized to simultaneously monitor both the conformational change of damage recognition proteins and distortion of the damaged DNA. In the third aim, we will attempt to functionally define the role of damage recognition factors in 3' and 5' incision activity by XPG and ERCCI-XPF will be examined. Both the accuracy and efficiency of the 3' and 5' incisions will be analyzed in advance with the kinetics of incision activity. Various mutants of damage recognition factors (RPA and XPC) will be used to examine any unique role these proteins possess in 3' and 5' incising.