reported the accumulation of a putative inter-
strand cross-link in DNA incubated withpks+
E. colias assessed by gel electrophoresis and
found that cell lines deficient in interstrand cross-
link repair were more sensitive to colibactin-
producingE. coli( 32 ).
To explore the correlation between DNA cross-
linking and the formation of 1 and 2 , we used a
modified alkaline single-cell gel electrophoresis
assay (comet assay) ( 33 ) to assess the presence
of interstrand cross-links in cells exposed to
pks+E. coli. This assay utilizes the high degree
of strand breaks induced bygradiation to mea-
sure interstrand cross-link formation. Unlike
monoadducts, interstrand cross-links inhibit
the denaturation of DNA under alkaline condi-
tions and therefore decrease the level of DNA
migration, reducing the ability to detect radiation-
induced strand breaks. We first tested whether
CometChip, a high-throughput platform and more
robust version of the comet assay ( 34 , 35 ), could
detect interstrand cross-links generated by cis-
platin, a bifunctional cross-linking agent. Cells
were exposed to varying concentrations (0 to
200 mg/ml) of cisplatin and then analyzed for
cross-links 6 hours after drug treatment. As
expected, cisplatin caused a significant decrease
in DNA migration in treated cells, thus confirm-
ing the utility of this assay (fig. S57).
Next, we applied CometChip to investigate
whether interstrand cross-links are formed in
HeLa cells exposed to colibactin at the same
time point at which we detected adducts 1 and 2.
HeLa cells were infected withpks+E. colifor
1 hour, and cross-link formation was measured
immediately afterward. Inpks+E. coli–treated
HeLa cells exposed togirradiation [8 gray (Gy)],
we detected a significant level of cross-links as
indicated by the 32% decrease in DNA tail mo-
ment compared to both controls (Fig. 4, B and C).
By contrast, we observed minimal strand breaks
in non-g-irradiatedpks+E. coli–treated cells.
Thus, these results indicate that interstrand cross-
links are present in thepks+E. coli–treated HeLa
cells from which we isolated 1 and 2. However,
we could not identify any masses corresponding
to putative interstrand cross-links in our untar-
geted DNA adductomics datasets, suggesting that
theymaybeunstabletoourisolation,purification,
or MS conditions.
Possible origins of colibactin-derived
DNA adducts
Knowledge of colibactin biosynthesis suggests
amechanismforhowDNAadductsorcross-links
degrade to form 1 and 2. Bioinformatic analyses
of the NRPS-PKS assembly line and isolation of
precolibactins have indicated that colibactin like-
ly contains a bithiazole ( 13 , 14 ) and/or a related
ring system with ana-aminoketone inserted be-
tween two thiazole rings (Fig. 1B) ( 15 ). However,
searching our untargeted DNA adductomics
datasets did not reveal masses corresponding to
putative bithiazole ora-aminoketone–containing
colibactin-DNA adducts. Because adducts 1 and
2 contain only one thiazole heterocycle, we pro-
pose that they derive from oxidative C–C cleavage
of a larger,a-aminoketone–containing colibactin-
DNA monoadduct or cross-link (Fig. 4A). Where-
as bithiazole rings are stable,a-aminoketones
undergo oxidative C–Cbondcleavageinthepres-
ence of reactive oxygen species to give carboxylic
acids ( 36 ). Therefore, the structures of 1 and 2
strongly suggest that the active genotoxin contains
ana-aminoketone, a positively charged functional
group that may enhance colibactin’s affinity for
DNA and thus increase its potency ( 22 ). Further
experiments will be needed to clarify the nature
of the interstrand cross-link, the origin and tim-
ing of the proposed oxidative degradation event,
and how the specific lesions(s) generated by co-
libactin lead to the formation of DBSs.Conclusions
We have presented direct evidence that the gut
bacterial genotoxin colibactin alkylates DNA
in vivo. The ability ofpks+E. colito generate
DNA adducts in mammalian cells and in mice
strengthens support for the involvement of
colibactin in cancer development or progression
because misrepaired monoadducts and cross-
linked adducts may generate mutations in onco-
genes or tumor suppressor genes, contributing to
tumorigenesis ( 37 , 38 ).Our findings will enable
efforts to decipher the molecular details of this
process. Importantly, this work has also uncov-
ered a candidate metabolite biomarker of coli-
bactin exposure and cancer risk. The ability to
directly assess whether exposure to colibactin has
occurredinanimalmodelsandhumanpatients
will help address the critical question of whether
pks+E. colicontribute to colorectal carcinogenesis
in patient cohorts ( 39 ). Finally, this study show-
cases the use of untargeted DNA adductomics for
the identification and elucidation of unknown
gut microbial-derived DNA modifications, high-
lighting the power of emerging analytical tech-
niques in studying human microbiota metabolites
and host-microbiota interactions.Materials and methods summary
Our methods for the preparation ofE. colistrains
for cell infection and isotope labeling, bacterial
monocolonization of germ-free mice, identifica-
tion of colibactin-derived DNA adducts by DNA
adductomics, synthetic preparation of model coli-
bactins and synthetic standards of the colibactin-
derived DNA adduct, structural characterization
of compounds by one- and two-dimensional NMR
methods and DP4 computational analysis, and
assessment of interstrand cross-link formation
using the CometChip assay are provided in the
supplementary materials. Additional informa-
tion about our protocols, including references
to the supplementary materials, can be found
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