title: Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I.
abstract: The effects of base sequence, specifically different pyrimidines flanking a bulky DNA adduct, on translesional synthesis in vitro catalyzed by the Klenow fragment of Escherichia coli Pol I (exo(-)) was investigated. The bulky lesion was derived from the binding of a benzo[a]pyrene diol epoxide isomer [(+)-anti-BPDE] to N(2)-guanine (G*). Four different 43-base long oligonucleotide templates were constructed with G* at a site 19 bases from the 5'-end. All bases were identical, except for the pyrimidines, X or Y, flanking G* (sequence context 5'-.XGY., with X, Y = C and/or T). In all cases, the adduct G* slows primer extension beyond G* more than it slows the insertion of a dNTP opposite G* (A and G were predominantly inserted opposite G, with A > G). Depending on X or Y, full lesion bypass differed by factors of approximately 1.5-5 ( approximately 0.6-3.0% bypass efficiencies). A downstream T flanking G on the 5'-side instead of C favors full lesion bypass, while an upstream C flanking G* is more favorable than a T. Various deletion products resulting from misaligned template-primer intermediates are particularly dominant ( approximately 5.0-6.0% efficiencies) with an upstream flanking C, while a 3'-flanking T lowers the levels of deletion products ( approximately 0.5-2.5% efficiencies). The kinetics of (1) single dNTP insertion opposite G* and (2) extension of the primer beyond G* by a single dNTP, or in the presence of all four dNTPs, with different 3'-terminal primer bases (Z) opposite G* were investigated. Unusually efficient primer extension efficiencies beyond the adduct (approaching approximately 90%) was found with Z = T in the case of sequences with 3'-flanking upstream C rather than T. These effects are traced to misaligned slipped frameshift intermediates arising from the pairing of pairs of downstream template base sequences (up to 4-6 bases from G*) with the 3'-terminal primer base and its 5'-flanking base. The latter depend on the base Y and on the base preferentially inserted opposite the adduct. Thus, downstream template sequences as well as the bases flanking G* influence DNA translesion synthesis.
authors.affiliation: Chemistry Department, New York University, 31 Washington Place, New York, New York 10003-5180, USA.
authors.forename: P
authors.initials: P
authors.lastname: Zhuang
authors.forename: A
authors.initials: A
authors.lastname: Kolbanovskiy
authors.forename: S
authors.initials: S
authors.lastname: Amin
authors.forename: N E
authors.initials: NE
authors.lastname: Geacintov
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002274:Carcinogens, Environmental / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis; D004249:DNA Damage / Q000235:genetics; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers / Q000302:isolation & purification / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D003849:Deoxyguanosine / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D009153:Mutagens / Q000737:chemistry / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods; D013698:Templates, Genetic; D013942:Thymine Nucleotides / Q000378:metabolism
chemical_list: D002274:Carcinogens, Environmental; D017931:DNA Primers; D003838:Deoxyadenine Nucleotides; D003845:Deoxycytosine Nucleotides; D003848:Deoxyguanine Nucleotides; D009153:Mutagens; D011089:Polydeoxyribonucleotides; D011742:Pyrimidine Nucleotides; D013942:Thymine Nucleotides; C071143:benz(a)pyrene-7,8-dihydrodiol-9,10-epoxide poly((dT-dC).(dG-dA)) adduct; C024107:2'-deoxycytidine 5'-triphosphate; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D006147:Guanine; C029603:deoxyguanosine triphosphate; D004256:DNA Polymerase I; D003849:Deoxyguanosine; C026600:2'-deoxyadenosine triphosphate; C024157:thymidine 5'-triphosphate