title: The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-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 O(6)-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O(6)-methylguanine (O(6)-MeG) and O(6)-carboxymethylguanine (O(6)-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates ( BENZI: TP and BIM: TP) 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 O(6)-MeG over guanine in the template. Furthermore, addition of artificial nucleotide BENZI: was required for full-length DNA synthesis during bypass of O(6)-CMG. Selective incorporation of the artificial nucleotide opposite an O(6)-alkylguanine DNA adduct was verified using a novel 2',3'-dideoxy derivative of BENZI: TP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BENZI: TP opposite biologically relevant O(6)-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies.
authors.affiliation: Department of Health Sciences and Technology, ETH Zu¨rich, 8092 Zürich, Switzerland.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Wyss
authors.affiliation: Department of Health Sciences and Technology, ETH Zu¨rich, 8092 Zürich, Switzerland.
authors.forename: Arman
authors.initials: A
authors.lastname: Nilforoushan
authors.affiliation: Center for Chemical Biology, Department of Chemistry, Krebs Institute, University of Sheffield, Sheffield S3 7HF, UK.
authors.forename: David M
authors.initials: DM
authors.lastname: Williams
authors.affiliation: Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, 78457 Konstanz, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Marx
authors.affiliation: Department of Health Sciences and Technology, ETH Zu¨rich, 8092 Zürich, Switzerland sturlas@ethz.ch.
authors.forename: Shana J
authors.initials: SJ
authors.lastname: Sturla
mesh_terms: D020029:Base Pairing / Q000235:genetics; D002273:Carcinogens / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D018736:DNA Adducts / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism*; D015203:Reproducibility of Results; D019914:Taq Polymerase / Q000378:metabolism; D013698:Templates, Genetic
chemical_list: D002273:Carcinogens; D018736:DNA Adducts; D009711:Nucleotides; C064976:O(6)-benzylguanine; D006147:Guanine; D004247:DNA; C008449:O-(6)-methylguanine; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase