Understanding the role of PCNA in DNA mismatch repair subpathways

了解 PCNA 在 DNA 错配修复子通路中的作用

• 批准号: 8698171
• 负责人: Eva Marie Goellner
• 金额: $ 5.33万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8698171
• 关键词: Affect; Alleles; Amino Acid Substitution; Base Pair Mismatch; Binding; Biochemical; Biological; Biological Assay; Boxing; Cells; Chemotherapy-Oncologic Procedure; Collection; Complex; Coupled; Coupling; Critical Pathways; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Data; Defect; Development; Discrimination; Dissociation; EXO1 gene; Escherichia coli; Event; Excision; Exodeoxyribonuclease I; Functional disorder; Future; Gene Mutation; Genes; Genetic; Genetic Screening; Genome Stability; Hereditary Malignant Neoplasm; Hereditary Nonpolyposis Colorectal Neoplasms; Human; Immunoprecipitation; In Vitro; Lead; Libraries; Light; MSH2 gene; MSH6 gene; Malignant Neoplasms; Maps; Minor; Mismatch Repair; Modeling; Molecular Sieve Chromatography; Mutation; Pathway interactions; Peptides; Phenotype; Plasmids; Play; Predisposition; Proliferating Cell Nuclear Antigen; Protein Binding; Proteins; Proto-Oncogenes; Reaction; Recruitment Activity; Resistance; Role; Saccharomyces cerevisiae; Signal Transduction; Site; Structure; Surface Plasmon Resonance; Syndrome; Techniques; Testing; Tumor Suppressor Genes; Yeasts; base; cancer genetics; chemotherapeutic agent; chemotherapy; in vivo; insertion/deletion mutation; mutant; prevent; public health relevance; reconstitution; repaired; research study; response; screening; spleen exonuclease; therapy development; tumor; yeast genetics

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.

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（进而驱动癌症的发展）至关重要。鉴于许多化疗药物通过损伤 DNA 发挥作用，了解 DNA 修复途径也至关重要。这样，DNA修复途径不仅影响癌症易感性，还影响癌症化疗的疗效。 DNA 错配修复 (MMR) 途径的作用是修复正常 DNA 复制过程中发生的碱基对错配和小插入/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。与明确的大肠杆菌 MMR 通路不同，真核 MMR 错配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种​​ MMR 子途径：1）外切核酸酶 1 (Exo1) 独立途径，似乎与 DNA 复制耦合；2) Exo1 依赖途径。人们对这些 MMR 亚通路的具体机制或其对人类癌症发展或化疗反应的影响知之甚少。 PCNA（增殖细胞核抗原）是 MMR 途径的一个组成部分，尽管其机制作用尚不完全清楚。 PCNA 是错配切除后 DNA 合成所必需的，并且在可能决定 MMR 亚途径功能的上游修复步骤中发挥多种作用。该项目将在编码 PCNA 的 POL30 中产生一系列功能分离突变，通过使用靶向遗传筛查，这些突变会导致 MMR 的 Exo1 独立或 Exo1 依赖的子通路功能障碍。这些突变将通过功能性生化和细胞生物学测定进行表征，以剖析 PCNA 在每个子通路中的机制作用，并阐明错配识别下游真核 MMR 的鲜为人知的细节。该机制数据将用于指导未来的实验，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。