Mechanism of DNA Damage Recognition in Higher Eukaryotes

顺铂是一种广泛使用的抗癌化疗药物，它通过在细胞中形成顺铂-DNA绑架物来诱导DNA损伤。在体内和在香橼的研究强烈表明，大多数顺铂-DNA绑架修复通过核苷酸切除修复（NER）途径。由于广泛的努力，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损伤是修复早期阶段的关键步骤。着色性干皮病A组互补蛋白（XPA）、复制蛋白A（RPA）、XPC-hHR 23 B和XPE可以独立地结合受损的DNA。然而，受损的识别蛋白如何在受损的DNA位点发挥作用仍存在争议。在本研究中，我们将使用生物化学和分子生物学的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的DNA位点发挥作用？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，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-hHR 23 B和TFIIH）和顺铂诱导的链内交联DNA分析单个损伤识别蛋白与损伤DNA的结合动力学，损伤识别因子之间的相互作用，以及切割前复合物的组装。在第二个目标中，我们将分析顺铂损伤的DNA的结构论文的分子基础。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。我们将使用足迹分析损伤识别因子诱导的损伤DNA的结构畸变。此外，荧光共振能量转移（FRET）方法将用于同时监测损伤识别蛋白的构象变化和受损DNA的畸变。在第三个目标中，我们将尝试通过XPG和ERCCI-XPF功能性地定义损伤识别因子在3'和5'切口活动中的作用。3'和5'切口的准确性和效率将通过切口活动的动力学提前分析。损伤识别因子（RPA和XPC）的各种突变体将用于检查这些蛋白质在3'和5'切割中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.