title: Benzo[a]pyrene-dG adduct interference illuminates the interface of vaccinia topoisomerase with the DNA minor groove.
abstract: Vaccinia DNA topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a pentapyrimidine target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrow in duplex DNA. The enzyme engages the target site within a C-shaped protein clamp. Here we mapped the interface of topoisomerase with the DNA minor groove by introducing chiral C-10 R and S 7,8-diol 9,10-epoxide adducts of benzo[a]pyrene (BP) at single N(2)-deoxyguanosine (dG) positions within the nonscissile DNA strand. These trans opened BPdG adducts fit into the minor groove without perturbing helix conformation or base pairing, and the R and S diastereomers are oriented in opposite directions within the minor groove. We measured the effects of the BPdG adducts on the rate and extent of single-turnover DNA transesterification. We observed a sharp margin of interference effects, whereby +5 and -2 BPdG modifications were well tolerated but +4, +3, and -1 BPdG adducts were severely deleterious. Stereoselective effects at the -1 nucleoside (the R isomer interfered, whereas the S isomer did not) delineated at high resolution the downstream border of the minor groove interface. BPdG inhibition of transesterification is likely caused by steric exclusion of constituents of the topoisomerase from the minor groove. We also applied the BPdG interference method to probe the interactions of exonuclease III with the minor groove. DNAs containing these BPdG adducts were protected from digestion by exonuclease III, which was consistently arrested at positions 2-4 nucleotides prior to the BP-modified guanosine.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: Ligeng
authors.initials: L
authors.lastname: Tian
authors.forename: Jane M
authors.initials: JM
authors.lastname: Sayer
authors.forename: Heiko
authors.initials: H
authors.lastname: Kroth
authors.forename: Govind
authors.initials: G
authors.lastname: Kalena
authors.forename: Donald M
authors.initials: DM
authors.lastname: Jerina
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D001483:Base Sequence; D001564:Benzo(a)pyrene / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D018736:DNA Adducts / Q000737:chemistry* / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D013997:Time Factors; D014615:Vaccinia / Q000201:enzymology*
chemical_list: D018736:DNA Adducts; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004264:DNA Topoisomerases, Type I