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\begin{document}

\title{The aflatoxin B\textsubscript{1}-induced imidazole ring-opened guanine
adduct: high mutagenic potential that is minimally affected by sequence
context}



\author[1]{Irina G. Minko}%
\author[2]{Andrew Kellum, Jr}%
\author[2]{Michael Stone}%
\author[1]{R. Stephen Lloyd}%
\affil[1]{Oregon Health and Science University}%
\affil[2]{Vanderbilt University Medical Center}%


\vspace{-1em}



  
  \date{May 19, 2024}


\begingroup
\let\center\flushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
Consumption of foods contaminated with aflatoxin B\textsubscript{1}
(AFB\textsubscript{1}) is a recognized risk factor for developing
hepatocellular carcinomas (HCCs). The mutational signature of
AFB\textsubscript{1} is characterized by high frequency G \textgreater{}
T transversions in a limited subset of trinucleotide sequences. The
8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin
B\textsubscript{1} (AFB\textsubscript{1}-FapyGua) has been implicated as
the primary DNA lesion responsible for AFB\textsubscript{1}-induced
mutations. This study evaluated the mutagenic potential of
AFB\textsubscript{1}-FapyGua in four contexts, including hot- and
cold-spot sequences as apparent in the mutational signature. Vectors
containing AFB\textsubscript{1}-FapyGua were replicated in primate cells
and the products of replication were isolated and sequenced. Regardless
of the sequence context, AFB\textsubscript{1}-FapyGua caused base
substitutions at frequencies of \textasciitilde{} 80-90\%, with G
\textgreater{} T transversions being most common. Spectra of mutations
were only slightly modulated by the sequence context. These data suggest
that mechanism(s) defining sequence context-dependent distribution of
AFB\textsubscript{1}-induced mutations likely operates prior to
replication.%
\end{abstract}%



\sloppy


\textbf{The aflatoxin B\textsubscript{1}-induced imidazole ring-opened
guanine adduct: high mutagenic potential that is minimally affected by
sequence context}

Irina G. Minko\textsuperscript{1}, Andrew H. Kellum,
Jr\textsuperscript{2}., Michael P. Stone\textsuperscript{2+}, R. Stephen
Lloyd\textsuperscript{1,3+}

\textsuperscript{1} Oregon Institute of Occupational Health Sciences,
Oregon Health \& Science University, 3181 SW Sam Jackson Park Rd.,
Portland, OR 97239

\textsuperscript{2} Department of Chemistry, Vanderbilt University,
Nashville, TN

\textsuperscript{3} Department of Molecular and Medical Genetics, Oregon
Health \& Science University, 3181 SW Sam Jackson Park Rd., Portland, OR
97239

\textsuperscript{+} Correspondence to: R. Stephen Lloyd, Oregon
Institute of Occupational Health Sciences, Oregon Health \& Science
University, L606, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA;
Tel: + 1 503 494 9957; Fax: + 1 503 494 4278; Email: lloydst@ohsu.edu

Running title: Mutagenic potential of the aflatoxin
B\textsubscript{1}-induced guanine adduct

Keywords: base substitution, hepatocellular carcinoma, replication
bypass, COSMIC signature SBS24

\textbf{Abstract}

Consumption of foods contaminated with aflatoxin
B\textsubscript{1}(AFB\textsubscript{1}) is a recognized risk factor for
developing hepatocellular carcinomas (HCCs). The mutational signature of
AFB\textsubscript{1} is characterized by high frequency G \textgreater{}
T transversions in a limited subset of trinucleotide sequences. The
8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin
B\textsubscript{1} (AFB\textsubscript{1}-FapyGua) has been implicated as
the primary DNA lesion responsible for AFB\textsubscript{1}-induced
mutations. This study evaluated the mutagenic potential of
AFB\textsubscript{1}-FapyGua in four contexts, including hot- and
cold-spot sequences as apparent in the mutational signature. Vectors
containing AFB\textsubscript{1}-FapyGua were replicated in primate cells
and the products of replication were isolated and sequenced. Regardless
of the sequence context, AFB\textsubscript{1}-FapyGua caused base
substitutions at frequencies of \textasciitilde{} 80-90\%, with G
\textgreater{} T transversions being most common. Spectra of mutations
were only slightly modulated by the sequence context. These data suggest
that mechanism(s) defining sequence context-dependent distribution of
AFB\textsubscript{1}-induced mutations likely operates prior to
replication.

\textbf{INTRODUCTION}

Several environmental mutagenic agents, including ultraviolet light,
components of tobacco smoke, aristolochic acid, and aflatoxin
B\textsubscript{1} (AFB\textsubscript{1}), are established human
carcinogens (Report on Carcinogens, National Toxicology Program, U.S.
Department of Health and Human Services, Public Health Service,
https://doi.org/10.22427/NTP-OTHER-1003). Following exposures of
cellular DNA, these agents or their metabolic byproducts induce distinct
mutational patterns that differ by the types of mutations, local
sequence contexts in which base substitutions preferentially occur, and
distribution of mutations throughout genome (Kucab et al. 2019;
Alexandrov et al. 2020; Volkova et al. 2020). Such agent-specific
mutational signatures are evident in deconstructed profiles of somatic
mutations in human cancers (Catalogue Of Somatic Mutations In Cancer
(COSMIC), https://cancer.sanger.ac.uk/signatures/) (Alexandrov et al.
2020).

AFB\textsubscript{1} is implicated as a significant risk factor for
developing hepatocellular carcinoma (HCC) (reviewed in (Kobets et al.
2022)). The most common route of human exposure to
AFB\textsubscript{1}is through consumption of food products contaminated
with\emph{Aspergillus flavus} and other related fungi producing this
toxin (reviewed in (Kensler et al. 2011)). Although the carcinogenic
effect of AFB\textsubscript{1} has been recognized for several decades,
such exposures remain widespread in many regions of Asia and sub-Saharan
Africa (Benkerroum 2020). As an example, recent analyses of somatic
mutations in 163 HCCs from Chinese patients demonstrated that
\textasciitilde{} 10\% of these cancers showed a typical signature of
AFB\textsubscript{1} (Zhang et al. 2017). The COSMIC data base reports
the AFB\textsubscript{1} footprint in 19 out of 493 HCCs (single base
substitution (SBS) signature 24)
(https://cancer.sanger.ac.uk/signatures/sbs/sbs24/).

Following ingestion, AFB\textsubscript{1} is metabolically activated by
microsomal enzymes to an epoxide, which can intercalate into DNA and
covalently bind to it, predominantly at the N7 position of guanine
(reviewed in (Kensler et al. 2011; McCullough and Lloyd 2019)). This
initial cationic\emph{trans}
-8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B\textsubscript{1}
(AFB\textsubscript{1}-N7-Gua) adduct is chemically unstable and can
either undergo hydrolytic base loss giving rise to an apurinic (AP)
site, or interact with a hydroxyl to produce the imidazole
ring-opened\emph{trans}
-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin
B\textsubscript{1} (AFB\textsubscript{1}-FapyGua) (\textbf{Figure 1} )
(Croy and Wogan 1981). Based on numerous observations summarized below,
the AFB\textsubscript{1}-FapyGua adduct is implicated as a major
contributor to AFB\textsubscript{1}-induced mutagenesis.

In contrast to the initial AFB\textsubscript{1}-N7-Gua adduct that
rapidly disappears from cellular DNA, AFB\textsubscript{1}-FapyGua
persists much longer. Specifically, while it comprised
\textasciitilde{}80\% of the AFB\textsubscript{1} adducts detected in
rat liver DNA 72 h post intraperitoneal injection (Croy and Wogan 1981),
in mice, it represented \textasciitilde{}95\% of the total
AFB\textsubscript{1} adducts at 48 h post injection (Vartanian et al.
2017; Coskun et al. 2019). Additionally, prior investigations, using
site-specifically modified DNA demonstrated that
AFB\textsubscript{1}-FapyGua was highly mutagenic in \emph{Escherichia
coli} and primate cells (reviewed in (McCullough and Lloyd 2019)). When
analyzed in primate (COS7) cells using a SV40-based shuttle vectors, it
induced \textasciitilde{}84\% G to T transversions, 8\% G to A
transitions, 2.5\% G to C transversions, and 2.5\% single nucleotide
deletions, with only \textasciitilde{}3\% of progeny vectors containing
no mutations at the target site (Lin et al. 2014). Furthermore, there is
a correlation between spectrum of AFB\textsubscript{1}-FapyGua-induced
base substitutions (Lin et al. 2014) and the mutagenic properties of
AFB\textsubscript{1}. These have been investigated by a variety of
methods, ranging from the assessment of mutations in vector DNA that was
pretreated with activated AFB\textsubscript{1} and replicated in cells,
to sequencing of genomic DNA isolated from exposed animals (reviewed in
(McCullough and Lloyd 2019)). Consistently throughout these
investigations, the data implicated DNA damage at guanines as a major
contributor to mutagenesis, with G \textgreater{} T transversions being
the predominant type.

Beyond the overall mutagenic G \textgreater{} T signature,
AFB\textsubscript{1}-induced base substitutions showed non-random,
sequence context-dependent distribution (Chawanthayatham et al. 2017;
Huang et al. 2017; Zhang et al. 2017; Fedeles and Essigmann 2018; Kucab
et al. 2019; Alexandrov et al. 2020; Volkova et al. 2020). When
frequencies of AFB\textsubscript{1}-induced base substitutions were
normalized against frequencies of trinucleotide sequences, the dominant
G \textgreater{} T transversions concentrated in the CGC and CGG
sequence contexts (Fedeles and Essigmann 2018). The molecular mechanisms
accounting for difference with respect to sequence contexts are
currently unknown. The present study addressed the question whether
mutational signature of AFB\textsubscript{1} can be explained by
differential, sequence-dependent fidelity of replication past
AFB\textsubscript{1}-FapyGua. Site-specifically modified vectors
containing this adduct in four different local sequence contexts were
constructed, replicated in COS7 cells, and progeny DNA analyzed for
mutations.

\textbf{MATERIAL AND METHODS}

\textbf{2.1 Preparation of AFB\textsubscript{1}-FapyGua-containing
oligodeoxynucleotides}

The 11-mer oligodeoxynucleotides site-specifically modified with
AFB\textsubscript{1}-FapyGua were prepared from unmodified
oligodeoxynucleotides containing the unique N7-guanine alkylation site
(5'-CCAT\emph{AGC} TACC-3', 5'-CCAT\emph{CGC} TACC-3', 5'-CCAT\emph{TGC}
TACC-3', or 5'-CCAT\emph{CGA} TACC-3', where the target and neighboring
sites are underlined). These were annealed with the corresponding 6-mer
oligodeoxynucleotides (5'-TAGCTA-3', 5'-TAGCGA-3', 5'-TAGCAA-3', and
5'-TATCGA-3') to form partially double-stranded DNA structures and
reacted with AFB\textsubscript{1} \emph{exo} -8,9-epoxide. The
conditions for chemical modifications, purification by reverse-phase
HPLC, and characterization by mass spectrometry were previously
described (Li et al. 2015; Tomar et al. 2021).

\textbf{2.2 Construction of single-stranded DNA vectors containing
site-specific AFB\textsubscript{1}-FapyGua}

To evaluate the mutagenic potential of AFB\textsubscript{1}-FapyGua in
different sequence contexts, we utilized the pSBL vector system, with
the AFB\textsubscript{1}-FapyGua-containing 11-mer oligodeoxynucleotides
inserted between the \emph{Eco} RV sites in a stem region of
single-stranded pSBL, using the same conditions as previously described
(Minko et al. 2017). Vectors were purified using Amicon Ultra 100K
centrifugal filters (Merck Millipore, LTD) with TE buffer (10 mM
Tris-HCl (pH 7.4) and 1 mM EDTA).

\textbf{2.3 Mutagenesis assays}

Transfection of COS7 cells with modified vectors, isolation of
replication products, and obtaining the individual clones using \emph{E.
coli} cells were performed according to the published procedures (Minko
et al. 2017). The plasmids were sequenced by DNA Sequencing Core, Vollum
Institute, Oregon Health \& Science University.

\textbf{2.4 Statistical Analyses}

The mean frequencies of base substitutions with standard errors were
calculated from three repeats of mutagenesis assays. To evaluate
statistical significance of the differences observed, the \emph{p}values
were calculated by the Student's \emph{t} -test, using KaleidaGraph 4.1
software (Synergy Software, Reading, PA).

\textbf{RESULTS AND DISCUSSION}

\textbf{3.1 Mutagenic properties of AFB\textsubscript{1}-FapyGua in
different sequence contexts}

The single-stranded pSBL vectors containing AFB\textsubscript{1}-FapyGua
in the AXC, CXC, TXC, and CXA (X = AFB\textsubscript{1}-FapyGua)
sequence contexts were constructed and replicated in COS7 cells. The CGC
sequence has been identified as a hot-spot for
AFB\textsubscript{1}-induced G \textgreater{} T transversions, while the
AGC, TGC sequences and especially the CGA sequence were less prone to
these substitutions (Fedeles and Essigmann 2018). Following isolation of
the progeny DNAs, individual clones were obtained by transformation
of\emph{E. coli} DH5\selectlanguage{greek}α \selectlanguage{english}cells and subjected to Sanger sequencing. The
experiments were conducted in triplicate, beginning from transfection of
COS7 cells. The sample size was fixed in advance at the level of ten
clones per DNA isolate, excluding clones that either did not contain the
insert sequences (\textasciitilde{} 20\%) or had alterations others than
point mutations (\textbf{Table 1} ).

These data revealed that regardless of the sequence contexts,
AFB\textsubscript{1}-FapyGua was highly mutagenic, inducing base
substitutions at frequencies of \textasciitilde{} 80-90\%
(\textbf{Figure 2} ). The difference between sequence contexts with
regard to non-mutagenic replication was insignificant. Similar to
results of the prior study, in which the TXA sequence was utilized (Lin
et al. 2014), mutational spectra were dominated by G \textgreater{} T
transversions. In addition, G \textgreater{} A transitions and G
\textgreater{} C transversions were observed in each of the four
sequences tested. The levels of G \textgreater{} A transitions were
higher in the AXC and CXA sequences relative to the CXC and TXC
sequences, with difference observed for the AXC versus CXC sequence
approaching significant level (\emph{p} = 0.057). The level of G
\textgreater{} C transversions was elevated in the CXA sequence;
relative to the AXC sequence, the difference was significant (\emph{p} =
0.047).

\textbf{3.2 Mutagenic potential of AFB\textsubscript{1}-FapyGua in
relation to mutational signature of AFB\textsubscript{1}}

High levels of base substitutions at the C/G sites, predominantly C/G
\textgreater{} A/T transversions, is a common feature of DNA isolated
from AFB\textsubscript{1}-exposed cells or animals and human
AFB\textsubscript{1}-driven cancers (COSMIC signature SBS24)
(Chawanthayatham et al. 2017; Huang et al. 2017; Zhang et al. 2017;
Fedeles and Essigmann 2018; Kucab et al. 2019; Alexandrov et al. 2020;
Volkova et al. 2020). AFB\textsubscript{1}-FapyGua has been implicated
as a major contributor to AFB\textsubscript{1}-induced mutagenesis and
carcinogenesis (reviewed in (McCullough and Lloyd 2019)) particularly,
based on observed correlation between the mutational spectra of
AFB\textsubscript{1} and the miscoding properties of
AFB\textsubscript{1}-FapyGua in replication bypass reactions (Lin et al.
2016) and cultured primate cells (Lin et al. 2014). However, a
limitation of these site-specific studies was that the analyses were
restricted to a single, TXA, local sequence context. Here, we
substantiated the hypothesis about a major role of
AFB\textsubscript{1}-FapyGua in AFB\textsubscript{1}-induced mutagenesis
and carcinogenesis by demonstrating its high potential for inducing base
substitutions, including high frequency G \textgreater{} T
transversions, in four additional sequence contexts (Figure 2).

The G \textgreater{} T transversions that constitute signature SBS24 are
not distributed randomly, but preferentially arise in a limited subset
of trinucleotide sequences (Chawanthayatham et al. 2017; Huang et al.
2017; Zhang et al. 2017; Fedeles and Essigmann 2018; Kucab et al. 2019;
Alexandrov et al. 2020; Volkova et al. 2020). Several mechanisms have
been considered through which this signature is manifested, including
sequence-dependent DNA adduct formation, stability of the initial
AFB\textsubscript{1}-N7-dG adduct, rates of repair, and fidelity of
replication bypass. The present data, in combination with previous
analyses (Lin et al. 2014), strongly suggest that differential fidelity
of replication bypass of AFB\textsubscript{1}-FapyGua does not account
for the occurrence of these mutational hot-spots since the adduct was
highly mutagenic in all five sequence contexts. The difference between
the hot-spot CGC sequence and three other sequences analyzed in the
present study was insignificant (Figure 2). Thus, mechanism(s) that
define sequence-dependent occurrence of AFB\textsubscript{1}-induced
mutations likely operates prior to replication. Consistent with such
possibility, we recently have demonstrated that sequence context
modulates the catalytic efficiency of human glycosylase NEIL1 acting on
AFB\textsubscript{1}-FapyGua, with rates of reactions being inversely
correlated with thermal stability of the adduct-containing DNA (Tomar et
al. 2021). We speculate that AFB\textsubscript{1}-FapyGua in a more
thermally-stable local environment would be more likely to escape repair
not only by NEIL1, but also by nucleotide excision repair and thus,
largely contribute to the final mutational signature of
AFB\textsubscript{1}. In support for a role of pre-replication events in
shaping the final mutational signature of AFB\textsubscript{1} is the
distribution of AFB\textsubscript{1}-induced mutations throughout
genome: these are enriched in the intergenic and late-replicating
regions and the signature has a transcriptional strand asymmetry
(https://cancer.sanger.ac.uk/signatures/sbs/sbs24/).

\textbf{3.3 Replication bypass of AFB\textsubscript{1}-FapyGua:
modulation by sequence context and proposed mechanisms}

Although AFB\textsubscript{1}-FapyGua was nearly equally mutagenic in
all sequence contexts tested, the spectra of mutations were partially
affected by the identities of neighboring nucleotides. Specifically,
there was a trend for elevated frequencies of G \textgreater{} A
transitions in the AXC sequence context. The frequency of G
\textgreater{} C transversions was increased in the CXA context. These
observations suggest that replication bypass of
AFB\textsubscript{1}-FapyGua can proceed via the primer-template
misalignment/realignment mechanism, with the 5' adjacent nucleotide
being utilized as a template instead of modified nucleotide. It is
possible that at least a fraction of G \textgreater{} T transversions
generated in either the TXC context or the previously tested TXA context
(Lin et al. 2014), is formed via a similar mechanism.

Our data demonstrate that even in the absence of a T at neighboring
positions, dAMP is preferentially inserted opposite
AFB\textsubscript{1}-FapyGua in primate cells. The mechanism accounting
for such a mutagenic event is currently unknown. It could be formation
of a mispair between the modified nucleotide and incoming dATP, as was
proposed for unsubstituted FapyGua (Gehrke et al. 2013), or
non-templated dAMP incorporation, according to the ``A rule'' (Strauss
2002), as previously proposed for \selectlanguage{greek}α-\selectlanguage{english}anomers of nitrogen mustard-induced
FapyGua (Minko et al. 2017). With regard to the later proposal,
AFB\textsubscript{1}-FapyGua exists as a complex mixture of various
isomeric species that include interconvertible \selectlanguage{greek}α- \selectlanguage{english}and \selectlanguage{greek}β-\selectlanguage{english}anomers (Brown
et al. 2009; Li et al. 2015). The fraction of \selectlanguage{greek}α-\selectlanguage{english}anomeric species is
substantial in single-stranded DNA (Brown et al. 2009).

The identity of DNA polymerase(s) responsible for bypass of
AFB\textsubscript{1}-FapyGua has been addressed by biochemical studies
with recombinant human DNA polymerases (Lin et al. 2014). The majority
of these enzymes could not synthesize DNA past
AFB\textsubscript{1}-FapyGua. The only polymerase that could, pol \selectlanguage{greek}ζ, \selectlanguage{english}did
it in an extremely error-prone manner by preferentially inserting a dAMP
opposite the adduct and extending exclusively from this mispair. Further
validation of the role of pol \selectlanguage{greek}ζ \selectlanguage{english}in the bypass of the
AFB\textsubscript{1}-FapyGua adduct was obtained from differential
cytotoxicity, cell cycle progression, and chromosomal abnormalities when
cells proficient or deficient in pol \selectlanguage{greek}ζ \selectlanguage{english}were challenged with the
AFB\textsubscript{1} epoxide (Lin et al. 2016).

\textbf{3.4 Mutagenic potential of AFB\textsubscript{1}-FapyGua: COS7
cells versus \emph{E. coli}}

Investigations on mutagenic properties of AFB\textsubscript{1}-FapyGua
in \emph{E. coli} utilized the CXA sequence context (Smela et al. 2002).
Comparison of these data with results of the present study demonstrated
dramatically lower fidelity of replication bypass of
AFB\textsubscript{1}-FapyGua in primate cells. While only three clones
out of 30 analyzed had no mutations in this sequence context in our data
set (\textbf{Figure 2} ), sequences with the correct G at the target
site were generated in \emph{E. coli} at frequencies of
\textasciitilde{}90\% under normal conditions or \textasciitilde{}60\%
following induction of SOS response.

\textbf{CONCLUSION}

The mutagenic properties of the major AFB\textsubscript{1}-induced
FapyGua adduct were assessed in primate cells using site-specifically
modified vectors with the adduct being positioned in four different
sequence contexts. Regardless of the context,
AFB\textsubscript{1}-FapyGua was highly mutagenic, predominantly causing
G \textgreater{} T transversions. These data provide further evidence
for a primary role of this adduct in AFB\textsubscript{1}-induced
mutagenesis. No correlation was apparent between the frequencies of
AFB\textsubscript{1}-FapyGua-induced G \textgreater{} T transversions
and the relative occurrences of these mutations in corresponding
trinucleotide sequences following AFB\textsubscript{1} exposure.
Collectively, these data suggest a role of pre-replicative events in
defining the mutational signature of AFB\textsubscript{1}.

\textbf{AUTHOR CONTRIBUTIONS}

Irina G. Minko: data collection and manuscript writing; Andrew H. Kellum
Jr.: preparation of modified oligodeoxynucleotides; Michael P. Stone and
R. Stephen Lloyd: study design and manuscript editing. All authors
edited the manuscript and approved its submission.

\textbf{ACKNOWLEDGEMENTS}

This work was supported by National Institutes of Health (NIH) grants
R01 CA-55678, R01 ES-029357, and P01 CA-160032 (M.P.S. and R.S.L.). The
Vanderbilt-Ingram Cancer Center is funded by NIH grant P30 CA-068485.
R.S.L. acknowledges support from the National Institute of Environmental
Health Sciences (R01 ES-031086) and from the Oregon Institute of
Occupational Health Sciences at Oregon Health \& Science University via
funds from the Division of Consumer and Business Services of the State
of Oregon (ORS 656.630). Funding for open access charge: NIH.

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\textbf{Table 1.} Complex mutation and targeted deletion in clones
isolated following replication of
AFB\textsubscript{1}-FapyGua-containing vectors in COS7 cells.\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
Sequence context\strut
\end{minipage} & \begin{minipage}[b]{0.47\columnwidth}\raggedright\strut
Identity of mutation\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
CXC\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Deletion starting at the -1 position relative to the lesion site:
5'-CCATCXCTACC-3' to 5'-(\textasciitilde{} 600 nt \selectlanguage{greek}Δ\selectlanguage{english})TACC-3'\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
CXC\strut
\end{minipage} & \begin{minipage}[t]{0.48\columnwidth}\raggedright\strut
Complex mutation: 5'-\emph{C}C\emph{AT}CX\emph{C}T\emph{A}CC-3' to
5'-\emph{G}C\emph{GG}CG\emph{A}T\emph{G}CC-3'\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{Figure legends}

\textbf{Figure 1.} Structure of AFB\textsubscript{1}-FapyGua.

\textbf{Figure 2.} Spectra of base substitutions induced by
AFB\textsubscript{1}-FapyGua following replication of site-specifically
modified vectors in COS7 cells. The data were collected from three
biological replicates. The uncertainties are standard errors.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Minko-et-al-Figure-1/Minko-et-al-Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Minko-et-al-Figure-2/Minko-et-al-Figure-2}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

