Recombination-based mechanisms for repair of damaged DNA replication templates

描述（由申请人提供）：DNA持续暴露于威胁整体基因组完整性的环境和内源性因素中。DNA损伤的一些主要因素是影响DNA单链的病变，或单模板病变（STLs）。这些有各种各样的表现，包括烷基化碱基、紫外线诱导的二聚体、单链刻痕和二级DNA结构（例如发夹和G4 DNA）。DNA复制机制绕过和修复这些病变的必要性是至关重要的，因为如果不这样做，可能会导致突变和不适当的重组，最终导致癌症或细胞衰老。迄今为止，许多不同的DNA修复途径已经在stl的旁路和修复中实现，包括五种同源重组（HR）依赖途径：双链断裂修复（DSBR）、断裂诱导复制（BIR）、叉反转（FR）、模板切换重组（TSR）和间隙修复（GR）。每一种途径都利用特定的重组中间体（RIs），前三种途径利用单或双Holliday结（HJs或dHJs），后两种途径利用Rec-Xs。本提案的目标是利用RIs的生化特征来确定哪些途径对特定类型的病变做出反应，以及哪些因素在这些途径中负责RIs的形成和消退。我的第一个具体目标是在酵母中使用遗传操作和药物治疗的组合来确定只有一种RI占主导地位的条件。我的第二个目标是利用新的合成DNA底物来确定对特定RIs的分辨率负责的因素。总之，这些目标将更好地理解许多hr通路如何共同作用以修复特定的stl，以及这些通路如何更好地靶向治疗癌症。

DESCRIPTION (provided by applicant): DNA is continuously exposed to environmental as well as endogenous factors that threaten overall genome integrity. Some of the primary contributors to DNA damage are lesions affecting one strand of the DNA, or single template lesions (STLs). These come in a variety of manifestations including alkylated bases, UV-induced dimers, single strand nicks, and secondary DNA structure (e.g. hairpins and G4 DNA). The necessity for DNA replication machinery to bypass and repair these lesions is of utmost importance, as failure to do so can result in mutagenesis and inappropriate recombination that ultimately leads to cancer or cellular senescence. To date, a number of different DNA repair pathways have been implemented in bypass and repair of STLs, including five Homologous Recombination (HR)-dependent pathways: Double Strand Break Repair (DSBR), Break Induced Replication (BIR), Fork Reversal (FR), Template Switch Recombination (TSR), and Gap Repair (GR). Each of these pathways employ specific recombination intermediates (RIs), with the former three pathways utilizing single or double Holliday Junctions (HJs or dHJs), and the latter two pathways utilizing Rec-Xs. The goal of this proposal is to use biochemical characterizations of RIs to determine which pathways respond to specific types of lesions, and which factors are responsible for the formation and resolution of RIs within these pathways. My first specific aim is to use a combination of genetic manipulation and drug treatment in yeast to determine conditions in which only one RI predominates. My second aim will utilize novel synthetic DNA substrates to identify factors responsible for the resolution of specific RIs. Together, these aims will provide a better understanding of how the many HR-pathways function together to repair specific STLs, and how these pathways can be better targeted to treat cancers.