Mechanisms of DNA interstrand crosslink unhooking and translesion synthesis

该项目将使用纯化蛋白系统和无细胞提取物来测试哺乳动物细胞中DNA链间交联（ICL）修复的两步模型。在G{1}期或静止期细胞中，模型结合了由核苷酸切除修复（NER）介导的脱钩步骤，在ICL的一条链的5'和3'上具有切口。关于这个问题，有相互矛盾的出版数据，本研究旨在解决。ICL修复模型的一个关键方面是可以以REV 3L依赖性方式绕过脱钩的ICL。这两种反应的检测将在细胞提取物中进行，包括那些在特定DNA修复因子中有缺陷的细胞。正常细胞提取物将被分级分离以确定哪些因子是完整反应所必需的，并且用纯化的蛋白质组分替代级分，以重现和重建这些过程。 具体目的1是通过在两侧切口来确定ICL的脱钩位置，并检查人细胞提取物的开放复合物形成。我们未发表的数据表明，哺乳动物细胞提取物可以切割优先作用于交联的吡喃酮侧的a的5'和3'。这一目标将与项目2合作实现。 具体目标2将确定ICL脱钩所需的蛋白质因子。实验将通过测试这些途径中具有特定缺陷的细胞系来检查反应的遗传依赖性。此外，提取物将被分级分离并与用纯化蛋白质重构的ICL脱钩。 具体目标3是测试ICL修复模型的最后阶段。我们将确定是否专门的DNA聚合酶可以绕过脱钩ICL和如何脱钩ICL可以在双链体DNA中删除。将使用细胞提取物和纯化的蛋白质来检查在未钩住的ICL上合成的遗传依赖性。待使用的蛋白质包括REV 3、REV 7、REV 1和其它DNA聚合酶。ICL修复的最后一步将被检查，使用代表连接到双链体DNA的未钩化的peptide交联的底物。将分析NER切割该底物的能力，并与BER切除进行比较。

This project will use purified protein systems and cell-free extracts to test models for two steps of DNA interstrand crosslink (ICL) repair in mammalian cells. In G{1} phase or quiescent cells, models incorporate an unhooking step mediated by nucleotide excision repair (NER), with incisions on one strand both 5' and 3' of an ICL. There is conflicting published data on this subject, which this research aims to resolve. A critical aspect of ICL repair models is that an unhooked ICL can be bypassed in a REV3L-dependent manner. Assays for both of these reactions will be carried out in cell extracts, including those defective in specific DNA repair factors. Normal cell extracts will be fractionated to determine which factors are necessary for the full reactions, and fractions substituted with purifed protein components, to recapitulate and reconstitute these processes. Specific Aim 1 is to define the positions of unhooking of an ICL by nicking on both sides, and examine open complex formation by human cell extracts. Our unpublished data indicates that mammalian cell extracts can cleave both 5' and 3' of a preferentially acting on the pyrone side of the crosslink. This aim will be carried out in collaboration with Project 2. Specific Aim 2 will identify the protein factors required for ICL unhooking. Experiments will examine the genetic dependence of the reaction by testing cell lines with specific defects in factors from these pathways. Further, extracts will be fractionated and unhooking of an ICL reconstituted with purified proteins. Specific Aim 3 is to test final stages of ICL repair models. We will determine whether specialized DNA polymerases can bypass an unhooked ICL and how an unhooked ICL can be removed in duplex DNA. The genetic dependence of synthesis over an unhooked ICL will be examined using cell extracts and purified proteins. Proteins to be employed include REV3, REV7, REV1 and other DNA polymerases. The final step in ICL repair will be examined, using a substrate representing an unhooked psoralen crosslink attached to duplex DNA. The ability of NER to cleave this substrate will be analyzed, and compared to excision by BER.