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  • AFB Fapy dG has been long recognized

    2022-05-18

    AFB1-Fapy-dG has been long recognized as a substrate for nucleotide excision repair [24,25]. However, we recently demonstrated that this lesion is removed by 961 excision repair. We showed that DNA glycosylase NEIL1, a member of the Fpg/Nei glycosylase family, could excise AFB1-Fapy-dG from site-specifically modified oligodeoxynucleotides in vitro and that mice accumulated significantly more of these lesions in liver DNA following a single AFB1 injection than mice. Furthermore, mice showed an increased susceptibility to AFB1-induced HCC versus control mice, implicating the AFB1-Fapy-dG adduct as a major contributor to AFB1-induced carcinogenesis [9]. In addition to AFB1-Fapy-dG, NEIL1 recognizes unsubstituted Fapy-dG and Fapy-dA, Me-Fapy-dG, thymine glycol (Tg), psoralen-induced adducts, and oxidation products of 8-oxo-deoxyguanosine, guanidinohydantoin and spiroiminohydantoin (Sp) [[26], [27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37]]. NEIL3, another member of the Fpg/Nei DNA glycosylase family, recognizes a broad class of lesions that significantly overlaps with that of NEIL1 [[37], [38], [39], [40], [41], [42]]. Germane to the present study, the shared substrates include unsubstituted Fapy-dG and Me-Fapy-dG. Thus, we hypothesized that NEIL3 would recognize AFB1-Fapy-dG and that both NEIL1 and NEIL3 would be able to initiate repair of NM-Fapy-dG. To address these possibilities, we measured the kinetics of product formation for the reaction of site-specifically modified oligodeoxynucleotides containing these or control lesions with human NEIL1 (hNEIL1) [32] or a truncated form of Mus musculus NEIL3 (MmuNEIL3Δ324) [38,42].
    Materials and methods
    Results and discussion
    Conflict of interest
    Acknowledgments We thank Dr. Sylvie Doublié (University of Vermont) for the kind gift of MmuNEIL3Δ324 and Dr. Carmelo Rizzo (Vanderbilt University) for insightful discussions and editing. This work was supported by the National Institutes of Health Grants P01 CA160032, P30 CA068485, R01 CA055678, and R01 ES029357.
    Introduction As a non-canonical base of DNA, uracil can occur in DNA by misincorporation of dUMP during DNA replication [1], [2] or spontaneous or enzymatic deamination of cytosine [3], [4], [5]. Uracil DNA glycosylase (UDG) is the principal enzyme to remove uracil in DNA by hydrolyzing the N-glycosidic bond and initiating base excision repair (BER) pathway [6], [7]. Based on the sequence homology and structural fold, six families were identified. Family 1 UDGs, also known as UNGs, are ubiquitous in bacteria and most eukaryotes. The enzymes exclusively cleave uracil in both double-stranded (ds) and single-stranded (ss) DNA at a very efficient rate [2], [8]. Family 2 UDGs are represented by human thymine DNA glycosylase (hTDG) [9] and E. coli mismatch-specific uracil DNA glycosylase (E. coli MUG) [10]. They are identified as repair enzymes acting on uracil base in a mispair, xanthine-containing DNA and other modified bases [10], [11], [12], [13], [14], [15]. The discovery of human TDG as a 5-formylcytosine (fC) and 5-caboxylcytosine (5-caC) DNA glycosylase places it as an essential enzyme in DNA demethylation [16], [17]. Family 3 enzymes are named as single-strand-selective monofunctional UDG (SMUG1) and are presented in vertebrate, insects and some eubacteria [18], [19]. Family 4 UDGs are a group of prokaryotic thermostable enzymes which excise uracil from DNA strand(s) [20]. Family 5 enzymes are also thermostable and present in archaea and eubacteria, with versatile substrates specificities [21]. Family 6 enzymes (HDG) recognize and excise hypoxanthine in DNA strand(s), exclusively [22]. Firstly isolated from Xenopus laevis and human, SMUG1 enzymes show few sequence homologies to other UDG families, but have similar gross structural fold [18]. Later, a genome database searching identified SMUG1 orthologs in a few eubacteria lacking family 1 UNG [19], [23]. In comparison to the robust family 1 UNG, SMUG1 enzymes are less active to uracil-containing DNA, but they are versatile with broader substrate specificity, in which they include uracil, xanthine, 5-formyluracil, 5-hydroxyuracil, 5-hydroxymethyluracil and 3,N4-ethenocytosine as substrate [19], [24], [25], [26], [27]. Depending on reaction conditions, SMUG1 presents different substrate preferences. Notably, in the presence of Mg2+ under physiological conditions, SMUG1 switches into double-stranded-selective UDG [28]. Through extensive biochemical and genetic analysis, SMUG1 was identified as the principal 5-formyluracil and 5-hydroxylmethyluracil DNA glycoyslase in mammalian systems [24], [26], [27], [29]. Later on, we reported that bacterial SMUG1, as well as human SMUG1, was not only a UDG, but also a xanthine DNA glycosylase [19]. The structures of the bacterial Geobacter metallireducens SMUG1 have been solved [30].