title: Intercalating polycyclic aromatic hydrocarbon-DNA adducts poison DNA religation by Vaccinia topoisomerase and act as roadblocks to digestion by exonuclease III.
abstract: Polycyclic aromatic hydrocarbon (PAH)-DNA adducts pervert the execution or fidelity of enzymatic DNA transactions and cause mutations and cancer. Here, we examine the effects of intercalating PAH-DNA adducts on the religation reaction of vaccinia DNA topoisomerase, a prototypal type IB topoisomerase (TopIB), and the 3' end-resection reaction of Escherichia coli exonuclease III (ExoIII), a DNA repair enzyme. Vaccinia TopIB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at a target site 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p / N(-1) in duplex DNA. The rate of the forward cleavage reaction is suppressed to varying degrees by benzo[a]pyrene (BP) or benzo[c]phenanthrene (BPh) adducts at purine bases within the 3'-G(+5)G(+4)G(+3)A(+2)A(+1)T(-1)A(-2) sequence of the nonscissile strand. We report that BP adducts at the +1 and -2 N6-deoxyadenosine (dA) positions flanking the scissile phosphodiester slow the rate of DNA religation to a greater degree than they do the cleavage rate. By increasing the cleavage equilibrium constant > or = 10-fold, the BPdA adducts, which are intercalated via the major groove, act as TopIB poisons. With respect to ExoIII, we find that (i) single BPdA adducts act as durable roadblocks to ExoIII digestion, which is halted at sites 1 and 2 nucleotides prior to the modified base; (ii) single BPhdA adducts, which also intercalate via the major groove, elicit a transient pause prior to the lesion, which is eventually resected; and (iii) BPh adducts at N2-deoxyguanosine, which intercalate via the minor groove, are durable impediments to ExoIII digestion. These results highlight the sensitivity of repair outcomes to the structure of the PAH ring system and whether intercalation occurs via the major or minor groove.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: Lyudmila
authors.initials: L
authors.lastname: Yakovleva
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Handy
authors.forename: Haruhiko
authors.initials: H
authors.lastname: Yagi
authors.forename: Jane M
authors.initials: JM
authors.lastname: Sayer
authors.forename: Donald M
authors.initials: DM
authors.lastname: Jerina
authors.forename: Stewart
authors.initials: S
authors.lastname: Shuman
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000737:chemistry / Q000506:poisoning*; D026942:DNA Topoisomerases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D007364:Intercalating Agents / Q000737:chemistry / Q000506:poisoning; D007700:Kinetics; D009690:Nucleic Acid Conformation; D011084:Polycyclic Aromatic Hydrocarbons / Q000737:chemistry / Q000506:poisoning*; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000201:enzymology*
chemical_list: D018736:DNA Adducts; D007364:Intercalating Agents; D011084:Polycyclic Aromatic Hydrocarbons; C095911:polycyclic aromatic hydrocarbons-DNA adduct; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D026942:DNA Topoisomerases