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Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the exo- Klenow fragment of Pol I.
- Source :
-
Biochemistry [Biochemistry] 2001 Jun 05; Vol. 40 (22), pp. 6660-9. - Publication Year :
- 2001
-
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.
- Subjects :
- 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide chemistry
Base Sequence
Carcinogens, Environmental chemistry
Carcinogens, Environmental metabolism
Catalysis
DNA Polymerase I chemistry
DNA Primers isolation & purification
DNA Primers metabolism
Deoxyadenine Nucleotides metabolism
Deoxycytosine Nucleotides metabolism
Deoxyguanine Nucleotides metabolism
Deoxyguanosine metabolism
Guanine metabolism
Kinetics
Mutagenesis
Mutagens chemistry
Mutagens metabolism
Polydeoxyribonucleotides chemistry
Pyrimidine Nucleotides metabolism
Sequence Analysis, DNA methods
Templates, Genetic
Thymine Nucleotides metabolism
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide metabolism
DNA Damage genetics
DNA Polymerase I metabolism
DNA Replication genetics
Polydeoxyribonucleotides metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 40
- Issue :
- 22
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 11380261
- Full Text :
- https://doi.org/10.1021/bi010005o