Understanding the role of PCNA in DNA mismatch repair subpathways

描述（由申请人提供）：DNA损伤修复途径对于维持基因组稳定性和防止原癌基因或肿瘤抑制基因突变（然后驱动癌症发展）至关重要。了解DNA修复途径也是至关重要的，因为许多化疗药物通过损伤DNA发挥作用。这样，DNA修复途径不仅影响癌症易感性，而且影响癌症化疗的疗效。DNA错配修复（MMR）途径的功能是修复正常DNA复制过程中发生的碱基对错配和小的插入/缺失错配。MMR缺陷导致突变率增加，并导致癌症易感综合征，如林奇综合征和散发性肿瘤。与定义明确的E. coliMMR途径中，真核MMR错配识别下游修复的确切机制尚不清楚。我们最近在S.酿酒酵母中发现了至少两种MMR子途径的存在：1）看起来与DNA复制偶联的外切核酸酶1（Exo 1）非依赖性途径和2）Exo 1依赖性途径。关于这些MMR子通路的具体机制或它们对人类癌症发展或对化疗反应的影响，我们知之甚少。PCNA（增殖细胞核抗原）是MMR通路的组成部分，尽管其机制作用尚未完全了解。PCNA是切除错配后DNA合成所必需的，并在上游修复步骤中发挥多种作用，可能决定MMR亚途径功能。该项目将通过使用靶向遗传筛选产生编码PCNA的POL 30中的功能分离突变的集合，这些突变导致MMR的Exo 1独立或Exo 1依赖的子途径功能障碍。这些突变的特点与功能的生化和细胞生物学分析，以剖析每个子通路内的PCNA的机制作用，并照亮了真核MMR下游的错配识别知之甚少的细节。这一机制数据将用于指导未来的实验，以了解PCNA突变和MMR亚通路缺陷如何影响人类肿瘤形成和化疗耐药性。

DESCRIPTION (provided by applicant): DNA damage repair pathways are critical for maintaining genome stability and preventing mutations in proto- oncogenes or tumor suppressor genes that then drive the development of cancer. Understanding DNA repair pathways is also critical given that many chemotherapeutic agents function by damaging DNA. In this way DNA repair pathways not only influence cancer susceptibility but also affect the efficacy of cancer chemotherapy. The DNA mismatch repair (MMR) pathway functions to repair base pair mismatches and small insertion/deletion mispairs that occur during normal DNA replication. Defects in MMR result in increased mutation rates and lead to cancer predisposition syndromes, such as Lynch syndrome, and sporadic tumors. Unlike the well-defined E. coli MMR pathway, the exact mechanisms of repair downstream of mispair recognition in eukaryotic MMR are still unclear. Our recent experiments in S. cerevisiae have uncovered the existence of at least two MMR sub pathways: 1) an Exonuclease1 (Exo1)-independent pathway that appears to be coupled to DNA replication and 2) an Exo1-dependent pathway. Little is known about the specific mechanisms of these MMR subpathways or their impact on human cancer development or response to chemotherapy. PCNA (Proliferating Cell Nuclear Antigen) is an integral part of the MMR pathway, although its mechanistic roles are not completely understood. PCNA is required for DNA synthesis after excision of the mispair, and plays multiple roles in upstream repair steps that potentially dictate MMR sub pathway function. This project will generate a collection of separation-of-function mutations in POL30, which encodes PCNA, that cause dysfunction in either the Exo1-independent or Exo1-dependent sub pathways of MMR by using targeted genetic screening. These mutations will be characterized with functional biochemical and cell biological assays to dissect the mechanistic roles of PCNA within each sub pathway and to illuminate the poorly understood details of eukaryotic MMR downstream of mispair recognition. This mechanistic data will be used to guide future experiments into how PCNA mutations and MMR sub pathway defects influence human tumor formation and chemotherapy resistance.