DOI: 10.5061/dryad.vmcvdnd5g
title: Data from: The specificity and structure of DNA crosslinking by the gut bacterial genotoxin colibactin

description: We report here LC-MS data, used in conjunction with NMR analysis, to
 elucidate the specificity and structure of interstrand crosslinks (ICL) of
 DNA resulting from the genotoxin colibactin. The measured masses of 14-
 and 25-mer double strand DNA oligomers exposed to colibactin provided for
 the determination of the chemical structure of colibactin-ICL. It revealed
 an α-ketoiminium in the central region of colibactin that likely serves as
 a key DNA recognition element, explaining colibactin’s sequence
 selectivity. A strand cleavage assay with high resolution LC-MS detection
 was used to study the site-specific products of various base sequences of
 25mer double-strand DNA oligomer sequences containing colibactin ICLs.
 This data was used to understand how and when colibactin-ICLs form.
description: # Data from: The specificity and structure of DNA crosslinking by the gut
 bacterial genotoxin colibactin Dataset DOI:
 [10.5061/dryad.vmcvdnd5g](https://doi.org/10.5061/dryad.vmcvdnd5g) ##
 Description of the data and file structure Data was acquired on Thermo
 Scientific Orbitrap Fusion and Lumos mass spectrometers and therefore the
 files are the vendors .raw data format. It can be processed directly using
 the vendors Xcalibur FreeStyle or Qualbrowser (older but still
 functional). An alternative free software which could be used is OpenChrom
 - an open-source alternative that can handle various mass spectrometry
 data formats. The samples with names containing "clb-" in the
 name are control samples which were exposed to e.coli which do not have
 the clb gene and therefore do not produce colibactin. The samples with
 "clb+" in the name are those which do produce colibactin. The
 data which was collected in triplicate has the each individual replicates
 indicated in its name so the first replicate has "Rep1" in the
 file name, the second replicate has "Rep2" in the file name, and
 the third replicate has "Rep3" in the file name. The varying
 part of the sequence is indicated in the file name. The data used to
 determine the mass of the colibactin interstrand crosslinked double strand
 14-mer oligomer (15-17 min retention time) is in the
 `Figure_3_14mer_ICL_Mass_Determination.zip` folder. The data used to
 determine the mass of colibactin interstrand crosslinked double strand
 25-mer oligomer (15.3-15.7 min retention time) is in the
 `Figure_S12_25mer_ICL_Mass_Determination.zip` folder. The data used to
 determine the masses of the 3-Deaza-dAdo cleavage samples (32.3-35.2 min
 retention time) is in the `Figure_S11_3-Deaza-dAdo_Cleavage.zip` folder.
 The data used to determine the masses of the AT-rich cleavage samples
 (32-35.2 min retention time) is in the `Figure_1_-_AT-rich_Cleavage.zip`
 folder. The data used to illustrate how the cleavage data analysis works
 (32-35.2 min retention time) is contained in the
 `Figure_S2_-_Cleavage_Assay_-_AAATT.zip` folder. The data used to
 determine the masses of the GC-containing cleavage samples (32.5-35.7 min
 retention time) is in the `Figure_S7_GC-containing_Cleavage.zip` folder.
 ***Oligonucleotide Data Analysis*** Oligonucleotide data analysis was
 performed using Thermo Scientific’s Protein Deconvolution and FreeStyle
 software packages and the online Mongo Oligo Mass Calculator tool.
 ***Cleavage Assay MS Data Analysis*** Identifications and relative
 abundance measurements of the base treatment-induced cleavage sites of the
 colibactin-exposed double strand oligonucleotides were made using
 isotopically-resolved charge-state mass deconvolution of their LC-HRAM-MS
 spectra. The molecular formulas of the base-treatment cleavage products
 are the same as the “w” and “d” product ions formed upon MS2 collisional
 induced dissociation (CID) of negatively charged unmodified single strand
 oligonucleotides (*64*). The “w” and “d” product ions for each single
 strand oligonucleotide were calculated online using the Mongo Oligo Mass
 Calculator v2.06
 ([http://mass.rega.kuleuven.be/mass/mongo.htm](http://mass.rega.kuleuven.be/mass/mongo.htm)) with the “CID fragments” feature and the  “monoisotopic mass”, “negative mode”, “DNA”, and the “5’-OH” and “3’-OH” terminals selected. Mass identities were verified by comparing to calculated masses of proposed structures in ChemDraw. The measured masses of the cleavage products were determined by deconvoluting the multiple charge states seen in full scan spectra acquired during the cleavage product retention period using Freestyle software (Thermo Scientific, Waltham, MA). The deconvoluted experimental masses from FreeStyle were compared to cleavage products masses calculated using the Mongo software (accounting for the charge state) to assign the cleavage products. The assigned cleavage products were then used to identify the location of the colibactin adduct of the intact oligonucleotide. Signal intensities from both the [M+H] and [M + Na] ions from each cleavage product were summed and normalized to the amount of DNA injected. Values plotted are the difference between the average signal intensities observed in assays with *pks*+ and *pks*– *E. coli*. ## Code/software Thermo Scientific Protein Deconvolution and Xcalibur FreeStyle and Qualbrowser software packages