Understanding the role of PCNA in DNA mismatch repair subpathways

描述(申请人提供)：DNA损伤修复途径对维持基因组稳定性和防止原癌基因或肿瘤抑制基因突变至关重要，这些基因突变进而推动癌症的发展。了解DNA修复途径也是至关重要的，因为许多化疗药物是通过破坏DNA来发挥作用的。通过这种方式，DNA修复途径不仅影响癌症的易感性，还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缺陷会导致突变率增加，并导致癌症易感综合征，如林奇综合征和散发性肿瘤。与已明确定义的大肠杆菌MMR途径不同，真核细胞MMR中错对识别下游修复的确切机制仍不清楚。我们最近在酿酒酵母中的实验发现，至少存在两条MMR亚途径：1)外切酶1(Exo1)不依赖于DNA复制的途径；2)Exo1依赖的途径。关于这些MMR亚通路的具体机制或它们对人类癌症发展或化疗反应的影响，人们知之甚少。增殖细胞核抗原(增殖细胞核抗原)是MMR途径的重要组成部分，但其作用机制尚不完全清楚。错配对切除后，DNA合成需要增殖细胞核抗原，并在上游修复步骤中扮演多种角色，这些步骤可能决定MMR亚途径的功能。该项目将通过有针对性的基因筛查，在编码增殖细胞核抗原的POL30中产生一系列功能分离突变，导致MMR的Exo1独立或依赖于Exo1的亚通路功能障碍。这些突变将通过功能生化和细胞生物学分析来表征，以剖析增殖细胞核抗原在每个亚途径中的机制作用，并阐明错对识别下游真核细胞MMR的鲜为人知的细节。这些机制数据将被用来指导未来的实验，即增殖细胞核抗原突变和MMR亚途径缺陷如何影响人类肿瘤的形成和化疗耐药性。

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.