A101555A_nrreporting-summary_2020_ 04_22 updated.pdf A. Additional Supplementary Files Type Number If there are multiple files of the same type this should be the numerical indicator. i.e. "1" for Video 1, "2" for Video 2, etc. Filename This should be the name the file is saved as when it is uploaded to our system, and should include the file extension. i.e.: Smith_ Supplementary_Video_1. mov Legend or Descriptive Caption Describe the contents of the file Supplementary Table 1 Tables.xlsx Supplementary Tables 1-2 Colibactin DNA damage signature indicates mutational impact in colorectal cancer
27Colibactin, a potent genotoxin of Escherichia coli, causes DNA double strand breaks (DSBs) in human 28 cells. We investigated if colibactin creates a particular DNA damage signature in infected cells by 29identifying DSBs in colon cells after infection with pks+ E.coli. Interestingly, genomic contexts of DSBs 30 were enriched for AT-rich penta-/hexameric sequence motifs, exhibiting a particularly narrow minor 31 groove width and extremely negative electrostatic potential. This corresponded with the binding 32 characteristics of colibactin to double-stranded DNA, as elucidated by docking and molecular dynamics 33 simulations. A survey of somatic mutations at the colibactin target sites of several thousand cancer 34 genomes revealed significant enrichment of the identified motifs in colorectal cancers. Our work 35 provides direct evidence for a role of colibactin in the etiology of human cancer. 36One sentence summary: We identify a mutational signature of colibactin, which is significantly 37 enriched in human colorectal cancers. 38 39 3 40The mucosal epithelium is a preferred target of damage by chronic bacterial infections and associated 41 toxins. Not surprisingly, most cancers originate from this tissue. Several infectious agents have been 42 implicated in human cancers, with Helicobacter pylori representing the prototype of a cancer-inducing 43bacterium. Yet, unlike for infections with tumor viruses, which deposit telltale transforming genes in 44 infected cells, for bacterial pathogens compelling evidence of a carcinogenic function is missing due to 45 the lack of specific signatures of past infections in the emerging cancer genomes. Nonetheless, a 46 broader role of bacterial pathogens in human carcinogenesis is highly suggestive. 47 exact sites of DSBs in fixed host cells 8 . The resulting next-generation sequencing (NGS) data and 65 computational analyses revealed a highly specific DNA damage signature, involving AT-rich sequence 66 patterns associated with extreme shape characteristics, which was confirmed by in silico modelling of 67 the colibactin interaction with DNA. By using this information for a stringent search of a mutational 68 signature of colibactin in human cancer genome data, we establish a role of colibactin in the cause of 69 human colorectal cancer and possibly additional cancer types. 70 71 An unbiased sequencing approach to detect colibactin-induced DSB patterns 72To confirm colibactin-induced damage, we infected the human colorectal adenocarcinoma cell line 73Caco-2 with pks+ E. coli at MOI 20 for 3 hours. Fluorescence immunohistochemistry showed that cells 74 infected with the wild-type bacteria (pks+) were positive for the DNA damage marker γH2AX, while 75 cells infected with the clbR deletion mutant (pks-), in which colibactin synthesis is restricted 9 , were 76 not ( Fig. 1A). To specifically gain insight into colibactin-induced DSBs and the DNA sequences at which 77 they occur, BLISS was applied to Caco-2 cells infected with WT M1/5 (pks+) and mutant ΔclbR M1/5 78 (pks-) E. col...
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