The use of an artificial nucleotide for polymerase-based recognition of carcinogenicO⁶-alkylguanine DNA adducts

Abstract

Enzymatic approaches for locating alkylation adducts at single-base resolution in DNA could enable new technologies for understanding carcinogenesis and supporting personalized chemotherapy. Artificial nucleotides that specifically pair with alkylated bases offer a possible strategy for recognition and amplification of adducted DNA, and adduct-templated incorporation of an artificial nucleotide has been demonstrated for a model DNA adduct O6-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O6-methylguanine (O6-MeG) and O6-carboxymethylguanine (O6-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2′-deoxynucleoside-5′-O-triphosphates (BenziTP and BIMTP) by an engineered KlenTaq DNA polymerase. The enzyme catalyzed specific incorporation of the artificial nucleotide Benzi opposite adducts, with up to 150-fold higher catalytic efficiency for O6-MeG over guanine in the template. Furthermore, addition of artificial nucleotide Benzi was required for full-length DNA synthesis during bypass of O6-CMG. Selective incorporation of the artificial nucleotide opposite an O6-alkylguanine DNA adduct was verified using a novel 2′,3′-dideoxy derivative of BenziTP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BenziTP opposite biologically relevant O6-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies.

Structural basis for the selective incorporation of an artificial nucleotide opposite a DNA adduct by a DNA polymerase

“…13 The effect mainly relies on the lower K M for BenziTP (18 mM vs. 410 mM) as the k cat is almost the same for BenziTP and dTTP (15.6 min À1 vs. 13.2 min À1 ). The efficiency of BenziMP incorporation opposite O 6 -MeG is still lower compared to the natural incorporation of dCMP opposite dG (260 fold less efficient for M747K 14 ) The difference relies mainly on the higher K M of BenziTP while k cat is almost the same. Structural data would be highly beneficial for optimizing nucleotide design towards higher incorporation efficiency which is crucial for furthering the sequencing approach.…”

“…It was described that presence of Mn 2+ can lower the K M of nucleotides binding to a DNA polymerase 18 which might aid to form a stable complex since the K M of BenziTP opposite O 6 -MeG is two orders of magnitude higher than the K M of the unmodified nucleotide in the same context (e.g. 18 mM for BenziTP opposite O 6 -MeG versus 0.7 mM for dCTP opposite dG as determined in 14 ). Ternary DNA polymerase complexes with manganese ions have already been obtained before and the position as well as the coordination patterns of the ions in the active sites were highly similar compared to the smaller magnesium ions.…”

“…1, BenziTP) opposite O 6 -methylguanine (O 6 -MeG) more efficiently than opposite guanine. 13,14 A mutated KlenTaq DNA polymerase was even more proficient and incorporated BenziMP up to 150-fold more efficiently opposite O 6 -MeG than opposite guanine. 13,14 The basis for the proficiency of the wildtype enzyme and the increased efficiency by the mutant DNA polymerase are not known, however, therefore limiting the knowledge needed to further engineer polymerase-based strategies for sequencing adducts.…”

“…The substrate BenziTP is bound in the active site and adopts an anti conformation. 14,22 It pairs with the O 6 -MeG templating nucleotide via two hydrogen bonds: one between the N1 of O 6 -MeG and the -NH donor on BenziTP (3.2 Å) and the second between the -NH 2 donor on O 6 -MeG and the carbonyl group of BenziTP (2.9 Å, Fig. 2).…”

N2‐Substituted 2′‐Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase κ

“…[7] Identification of nucleotide analogs as selective substrates or inhibitors for specific polymerases is challenging because all polymerases utilize the four cononocial dNTPs, and correct base pairing is mostly dependent on the polymerase recognizing Watson-Crick geometry. Recently, engineered polymerases were utilized to create a modifed nucleotide that can recognize a carcinogen-modified DNA template, [8] and expanded size dNTPs (dxNTPs) have been shown to have some selectivity for human DNA polymerase θ. [9] In this report, we utilize the known reactivity of DNA polymerase κ to rationally design N 2 -benzyl-dGTPs that are highly specific substrates for pol κ.…”