Base sequence dependence of in vitro translesional DNA replication past a bulky lesion catalyzed by the Exo- Klenow fragment of Pol I

P. Zhuang, A. Kolbanovskiy, S. Amin, N. E. Geacintov

Research output: Contribution to journalArticlepeer-review


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 N2-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′-...XG*Y..., 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 ∼1.5-5 (∼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 (∼5.0-6.0% efficiencies) with an upstream flanking C, while a 3′-flanking T lowers the levels of deletion products (∼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 ∼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.

Original languageEnglish (US)
Pages (from-to)6660-6669
Number of pages10
Issue number22
StatePublished - Jun 5 2001

ASJC Scopus subject areas

  • Biochemistry


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