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

基本信息

批准号：
10304123
负责人：
Erik S Carlson
金额：
$ 6.64万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
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/10304123
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 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

项目摘要

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：人类肠道菌群在健康和疾病中起着重要作用的证据正在稳步增长大肠杆菌与结直肠癌（CRC）的相关性的显着示例，癌症死亡的第二个主要原因。> 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+）编码为遗传毒性的天然产物，称为Colibactin 已知产生结肠癌的细菌会经历G2/M细胞周期停滞，衰老和大核细胞增多症并具有DNA双链断裂（DSB），链间交联（ICL）和染色体尺寸。由于这些原因，许多人推测结肠癌是theSesessess和Clb+ E的化学介体。大肠杆菌在CRC中起着至关重要的作用；很难，因为它从未被隔离，而是结构和生物活性通过研究其生物合成和DNA损害特性而慢慢地启发出来。结肠癌是“伪二聚体”的交联剂在体外和细胞中产生ICL。 ICL导致发现了两个DNA单aducts 该提案试图通过1）量化Colibactin- DNA加合物并将水平与已建立的CLB+大肠杆菌表型在体外和体内以及2 与DNA的成功压缩时，确定结肠癌的序列特异性和结构。 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生物标志物可以适用于癌症监测和预防。科比乳素的结合基序，可以应用于识别人类内的靶基因，并确定A 结构上的结构性解释是科里氏素如何损害DNA。科比氏素基因毒素的定量和机械证据，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。