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

项目总结： 人类肠道微生物区系在健康和疾病中发挥重要作用的证据正在稳步增加。一个 这方面的显著例子是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。&gt；60%的结直肠癌患者体内有携带CLB的大肠杆菌 生物合成基因簇(CLB+)，编码一种具有遗传毒性的天然产物，名为Colibactin。暴露的单元格 已知产生结肠素的细菌会经历G2/M期细胞周期停滞、衰老和巨噬细胞增多症 并具有DNA双链断裂(DSB)、链间交联(ICL)和染色体畸变。 出于这些原因，许多人推测，Colibactin是这些过程的化学媒介，而Clb+E。 Coli在结直肠癌中起着至关重要的作用；然而，确定Colibactin遗传毒性背后的化学机制已经 这是困难的，因为它从来没有被孤立过。相反，Colibactin的结构和生物活性 通过研究其生物合成和DNA损伤特性，慢慢揭示了这一点。这些研究表明， Colibactin是一种“伪二聚体”交联剂，可在体外和细胞内产生ICL。试图分离出 ICL导致发现了两个DNA单加合物，每个单加合物对应于原始交联键的一半。 这项建议试图通过1)量化Colibactin--来破译Colibactin的遗传毒性的化学机制。 DNA加合物及其水平与体内和体外已建立的Clb+大肠杆菌表型的相关性 确定与DNA结合时的结肠肌动蛋白的序列特异性和结构。成功完成这些任务 AIMS将提供专门测量Colibactin-DNA损伤和潜在Colibactin的新方法 可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 Colibactin的结合基序，可用于识别人类体内的靶基因，并确定 丛生肌动蛋白如何损伤和扰乱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理证据及其作用的进一步认识 Clb+大肠杆菌在癌症的发病机制中起着重要作用。

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.