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始致癌基因或肿瘤抑制基因的突变至关重要，然后促进癌症的发展。考虑到许多化学治疗剂通过损害DNA的功能，了解DNA修复途径也至关重要。通过这种方式，DNA修复途径不仅会影响癌症的敏感性，还会影响癌症化疗的功效。 DNA不匹配修复（MMR）途径的功能可修复基本对不匹配和在正常DNA复制过程中发生的小插入/缺失失误。 MMR的缺陷导致突变率增加并导致癌症易感综合征，例如林奇综合征和零星肿瘤。与定义明确的大肠杆菌MMR途径不同，真核MMR中MispAir识别下游修复的确切机制尚不清楚。我们最近在酿酒酵母中的实验发现了至少两个MMR子途径的存在：1）外核酸酶1（EXO1）独立途径似乎与DNA复制耦合，并且2）exo1依赖性途径。对于这些MMR子路的特定机制或它们对人类癌症发展或对化疗的反应，知之甚少。 PCNA（增殖细胞核抗原）是MMR途径不可或缺的一部分，尽管其机械作用尚未完全了解。 MispAir切除后DNA合成需要PCNA，并且在可能决定MMR子途径功能的上游修复步骤中扮演多个角色。该项目将在编码PCNA的POL30中产生一系列功能分离突变，该突变通过使用靶向遗传筛选，在exo1独立或依赖于EXO1依赖性或exo1依赖性子途径中引起功能障碍。这些突变将以功能性生化和细胞生物学测定为特征，以剖析PCNA在每个子途径中的机械作用，并阐明MispAir识别下游的真核MMR的知识细节。该机械数据将用于指导未来的实验，以了解PCNA突变和MMR子途径缺陷如何影响人类肿瘤的形成和化学疗法抗性。