Deciphering the mechanism of colibactin-induced DNA damage through quantitative and biochemical approaches

通过定量和生化方法解读大肠杆菌素诱导的 DNA 损伤机制

• 批准号: 10472642
• 负责人: Erik S Carlson
• 金额: $ 4.76万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-06 至 2023-02-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10472642
• 关键词: Alkylation; Anabolism; Animals; Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Biological Markers; Biological Models; Cancer Etiology; Cell Cycle; Cell Cycle Arrest; Cells; Cessation of life; Characteristics; Chemical Models; Chemicals; Chemistry; Chromosome abnormality; Colitis associated colorectal cancer; Colorectal Cancer; DNA; DNA Adducts; DNA Binding; DNA Damage; DNA Double Strand Break; DNA Interstrand Crosslinking; DNA Repair; DNA Sequence; Data; Disease; Early Diagnosis; Escherichia coli; Exposure to; Gene Cluster; Genes; Goals; Health; Home; Human; Human Cell Line; In Vitro; Inflammatory Bowel Diseases; Isotopes; Knowledge; Lead; Lesion; Link; M cell; Malignant Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Mediator of activation protein; Methods; Minor Groove; Modeling; Mus; Mutagens; Names; Natural Products; Oncogenes; Pathogenesis; Patients; Phenotype; Play; Prevention; Process; Property; Proteobacteria; Research; Role; Specificity; Stable Isotope Labeling; Structure; Techniques; Testing; Therapeutic Intervention; United States; Work; adduct; analog; biomarker development; cancer initiation; cancer prevention; chemical standard; chemical synthesis; colon cancer patients; colorectal cancer prevention; crosslink; cyclopropane; diagnostic biomarker; dimer; exposed human population; genotoxicity; gut bacteria; gut microbiota; in vivo; insight; interest; microorganism; novel; response; screening; senescence; specific biomarkers; tumorigenesis

Project Summary: Evidence for the human gut microbiota playing a significant role in health and disease is steadily growing. A notable example of this is the association of certain commensal strains of E. coli with colorectal cancer (CRC), the second leading cause of cancer deaths. >60% of CRC patients are home to E. coli possessing the clb biosynthetic gene cluster (clb+) which encodes for a genotoxic, natural product named colibactin. Cells exposed to colibactin-producing bacteria are known to undergo G2/M cell cycle arrest, senescence, and megalocytosis and possess DNA double-strand breaks (DSBs), interstrand crosslinks (ICLs), and chromosomal aberrations. For these reasons, many speculate that colibactin is the chemical mediator for these processes and that clb+ E. coli play a vital role in CRC; however, determining the chemical mechanism behind colibactin’s genotoxicity has been difficult because it has never been isolated. Instead, the structure and bioactivity of colibactin has been slowly revealed by studying its biosynthesis and DNA-damaging properties. These studies have revealed that colibactin is a ‘pseudo-dimeric’ crosslinking agent that produces ICLs in vitro and in cells. Attempts to isolate the ICL lead to the discovery of two DNA monoadducts, which each correspond to one-half of the original crosslink. This proposal seeks to decipher a chemical mechanism for colibactin’s genotoxicity by 1) quantifying colibactin- DNA adducts and correlating their levels to established clb+ E. coli phenotypes in vitro and in vivo and 2) determining colibactin’s sequence specificity and structure when bound to DNA. Successful completion of these aims will deliver novel methods for specifically measuring colibactin-DNA damage and potential colibactin biomarkers that could be applied to cancer surveillance and prevention. Additionally, this work will identify colibactin’s binding motif, which could be applied to identifying target genes within humans, and determine a structural explanation for how colibactin damages and perturbs DNA. Ultimately, this work will provide quantitative and mechanistic evidence for colibactin’s genotoxicity and a heightened understanding of the role clb+ E. coli plays in the pathogenesis of cancer.

项目摘要： 人类肠道微生物群在健康和疾病中起着重要作用的证据正在稳步增长。一个 明显的例子是大肠杆菌的某些共生菌株与结直肠癌（CRC）的关联， 癌症死亡的第二大原因。 > 60％的CRC患者是拥有CLB的大肠杆菌的家园 生物合成基因簇（CLB+）编码为遗传毒性的自然产物，称为Colibactin。细胞暴露 众所周知，产生结肠癌的细菌会经历G2/M细胞周期停滞，感应和大核细胞增多症 并具有DNA双链断裂（DSB），链间交联（ICL）和染色体畸变。 由于这些原因，许多人推测结肠癌是这些过程的化学介体，并且CLB+ E. 大肠杆菌在CRC中起着至关重要的作用；然而，确定科比乳素遗传毒性背后的化学机制具有 很难，因为它从未被隔离。相反，结肠癌的结构和生物活性已经 通过研究其生物合成和DNA损害特性而缓慢揭示。这些研究表明 结肠癌是一种“伪二聚体”交联剂，在体外和细胞中产生ICL。试图隔离 ICL导致发现了两个DNA单aducts，每个DNA单载体都与原始交叉链接的一半相对应。 该提案旨在通过1）量化Colibactin- DNA加合物并将其水平与已建立的CLB+大肠杆菌表型在体外和体内以及2 与DNA结合时，确定结肠癌的序列特异性和结构。这些成功完成 AIMS将提供新的方法，用于特异性测量Colibactin-DNA损伤和潜在的结肠癌 可以应用于癌症监测和预防的生物标志物。此外，这项工作将确定 科比素的结合基序，可以应用于识别人类内的靶基因，并确定一个 结构性解释了科里氏素如何损害DNA。最终，这项工作将提供 科比氏素的遗传毒性的定量和机械证据，并对角色有了更高的了解 CLB+大肠杆菌在癌症的发病机理中发挥作用。