br Acknowledgements This work was

Acknowledgements This work was supported by grants from la Ligue Nationale Française Contre le Cancer « Equipe LNCC 2016 » and Electricité de France RB 2016-17 (to M.S.), NU ORAU (http://www.nu.edu.kz) and Ministry of Education and Science of the Republic of Kazakhstan, program 0115RK03029 (to B.T.M.), the Federal Agency of Scientific Organizations VI.57.1.2/0309-2016-0001 (to N.A.K.), National Science Center in Poland, grant no. 2014/15/B/NZ5/01444 (to B.T.), and French National Center for Scientific Research (PRC CNRS/RFBR n1074 REDOBER) (to A.A.I.). ELC was supported by grants from CNano Ile de France, Plan Cancer Inserm, and Paris Sud University (MRM). The pre-steady-state kinetic studies of MBD4 were supported by the Russian Science Foundation grant # 16-14-10038 (to O.S.F.). O.K. and M.B. were supported by the fellowships from the Russian Foundation for Basic Research # 16-04-00037 and Bourses du Gouvernement Français program, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Introduction Cellular DNA is susceptible to attack by alkylating agents that could originate from either normal metabolic processes or as result of exposure to various environmental toxicants. The latter compounds include aflatoxin B1 (AFB1), the product of Aspergillus flavus and related fungi that contaminate food crops. Chronic dietary exposure to AFB1 represents an increased etiological risk for developing hepatocellular carcinoma (HCC) [1,2]. DNA alkylation is also induced by many chemotherapeutic drugs, such as temozolomide and nitrogen mustards (NMs), and is largely responsible for therapeutic effectiveness of these agents [3,4]. The guanine N7 kn to nm is the most nucleophilic site within DNA bases [5] and is the primary target of alkylation (Fig. 1). Initial N7-deoxyguanosine (N7-dG) adducts can undergo secondary reactions because of the positive charge on the imidazole ring, yielding either an abasic site or the -substituted formamidopyrimidine (Fapy-dG) adducts [6]. The alkyl-Fapy-dGs are more chemically stable than the corresponding N7-alkylated dGs, leading to much longer half-lives of the alkylated lesions in cellular DNA [[6], [7], [8], [9]]. The analyses of DNA modifications in livers of Fischer rats that were intraperitoneally injected with a single AFB1 dose demonstrated rapid disappearance of the original N7-dG adducts, with an apparent half-life of 7.5 h [8]. Approximately 20% of these adducts were converted to AFB1-Fapy-dGs that became the dominant lesions in 24 h and persisted during the 72-h period studied. When DNA repair-proficient mice were intraperitoneally injected with a single, non-lethal AFB1 dose, the AFB1-N7-dG and AFB1-Fapy-dG adducts were present in liver DNA 6 h post-injection at comparable levels, approximately 50 and 68 pmoles/mg, respectively [9]. By 48 h, the initial N7-dG adducts essentially disappeared, while the level of AFB1-Fapy-dGs was reduced less than 2-fold. Although kinetics of conversion have not been addressed in vivo for the NM-induced adducts, N7-dG and Fapy-dG modifications were detected in cultured cancer cells treated for 24 h with bis(2-chloroethyl)amine at the levels of 970 and 180 adducts per 107 nucleotides, respectively [7]. As expected, this bifunctional agent also induced the formation of DNA crosslinks, including both NM-N7-dG-N7-dG (240 per 107 nucleotides) and NM-Fapy-dG-N7-dG (6 per 107 nucleotides). Structural and biochemical investigations on Fapy-dG adducts are complicated by the fact that these adducts exist as a mixture of interconverting α- and β-anomeric forms (Fig. 1). The β species are predominant in double-stranded (ds) DNA. This has been shown for the temozolomide-induced methyl-Fapy-dG (Me-Fapy-dG) [10], NM-Fapy-dG [11], and AFB1-Fapy-dG [[12], [13], [14], [15]]. The N5-substituted Fapy-dG adducts manifest a strong mutator phenotype. As demonstrated by analyses of sequence alterations following replication of site-specifically modified vectors in primate (simian kidney COS7) cells, the spectra of mutations induced by these lesions were dominated by base substitutions, with G to T transversions being most common [[16], [17], [18]]. The overall mutation frequencies measured for Me-Fapy-dG ranged from ∼9 to 21%, depending on the local sequence context [16], and ∼11–12% for NM-Fapy-dG [18]. The AFB1-Fapy-dG adduct caused mutations at an unprecedentedly high ∼97% frequency, including ∼86% G to T transversions [17]. This spectrum parallels the mutational signature of AFB1 that was determined using DNA from exposed cultured cells or experimental animals, or from AFB1-associated HCCs [[19], [20], [21], [22], [23]].