Mechanisms of DNA interstrand crosslink unhooking and translesion synthesis

该项目将使用纯化的蛋白质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)修复的两个步骤的模型。在G{1}期或静止细胞中，模型包含一个由核苷酸切除修复(NER)介导的解钩步骤，在ICL的一条链上同时切开5‘和3’。关于这一主题，有相互矛盾的公开数据，这项研究旨在解决这一问题。ICL修复模型的一个关键方面是，可以以REV3L依赖的方式绕过未挂钩的ICL。对这两种反应的检测将在细胞提取液中进行，包括那些特定DNA修复因子缺陷的细胞提取液。正常的细胞提取物将被分级，以确定哪些因素是整个反应所必需的，并用纯化的蛋白质成分取代分级，以概括和重建这些过程。 具体目标1是确定ICL通过在两侧划痕来解钩的位置，并检查人类细胞提取物形成开放的复合体。我们未发表的数据表明，哺乳动物细胞提取物可以切割5‘和3’的A，优先作用于交联链的吡喃酮一侧。这一目标将与项目2合作实现。 具体目标2将确定ICL解钩所需的蛋白质因子。实验将通过测试在这些途径的因子中具有特定缺陷的细胞系来检查反应的遗传依赖性。此外，提取物将被分离，并与纯化的蛋白质重组的ICL脱钩。 具体目标3是测试ICL修复模型的最后阶段。我们将确定专门的DNA聚合酶是否可以绕过去挂钩的ICL，以及如何在双链DNA中去除去挂钩的ICL。我们将使用细胞提取液和纯化的蛋白质来研究非连接的ICL对合成的遗传依赖性。使用的蛋白质包括Rev3、Rev7、Rev1和其他DNA聚合酶。ICL修复的最后一步将被检查，使用的底物代表连接到双链DNA上的去钩补骨脂素交联物。将分析NER切割这一底物的能力，并将其与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 Our unpublished data indicates that mammalian cell extracts can cleave both 5' and 3' of a preferentially acting on the pyrone side of the crosslink.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.