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: 10065857
• 负责人: Erik S Carlson
• 金额: $ 6.49万
• 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 2023-07-05
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10065857
• 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; DNA Structure; Data; Disease; Early Diagnosis; Escherichia coli; Exposure to; Gene Cluster; Genes; Goals; Health; Home environment; 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发挥重要作用的证据正在稳步增长。一 这方面的一个显著的例子是某些大肠杆菌菌株的关联。大肠杆菌伴结直肠癌（CRC）， 癌症死亡的第二大原因。>60%的CRC患者是E.具有clb的大肠杆菌 生物合成基因簇（clb+），其编码称为大肠杆菌素的基因毒性天然产物。细胞暴露 已知大肠杆菌素产生菌经历G2/M细胞周期停滞、衰老和巨细胞增多 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，许多人推测大肠杆菌素是这些过程的化学介质，而clb+ E. 大肠杆菌在CRC中起着至关重要的作用;然而，确定大肠杆菌素遗传毒性背后的化学机制， 这很难，因为它从来没有被孤立过。相反，大肠杆菌素的结构和生物活性已经被研究。 通过研究它的生物合成和DNA损伤特性，这些研究表明， 大肠杆菌素是一种在体外和细胞中产生ICL的“假二聚体”交联剂。试图分离出 ICL导致两个DNA单加合物的发现，每个对应于原始交联的一半。 该提案试图通过以下方式破译大肠杆菌素遗传毒性的化学机制：1）定量大肠杆菌素， DNA加合物，并将其水平与建立的clb+ E相关联。大肠杆菌表型在体外和体内和2） 确定大肠杆菌素与DNA结合时的序列特异性和结构。成功完成这些 aims将提供专门测量大肠杆菌素-DNA损伤和潜在大肠杆菌素的新方法 这些生物标志物可用于癌症监测和预防。此外，这项工作将确定 colibactin的结合基序，可用于识别人体内的靶基因，并确定一个 大肠杆菌素如何破坏和干扰DNA的结构解释。最终，这项工作将提供 大肠杆菌素遗传毒性的定量和机制证据，以及对大肠杆菌素作用的进一步理解 clb+ E.大肠杆菌在癌症发病中的作用。