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

点击翻译按钮获取中文摘要

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.