Primer length dependence of binding of DNA polymerase I Klenow fragment to template-primer complexes containing site-specific bulky lesions

Olga Rechkoblit, Shantu Amin, Nicholas E. Geacintov

Research output: Contribution to journalArticle


The binding of the benzo[a]pyrene metabolite anti-BPDE (r7,t8-dihydroxy- t9,10-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene) to the N2 group of 2'- deoxyguanosine residues (dG*) is known to adversely affect the Michaelis- Menten primer extension kinetics catalyzed by DNA Pol I and other polymerases. In this work, the impact of site-specific, anti-BPDE-modified DNA template strands on the formation of Pol I (Klenow fragment, KF)/template-primer complexes has been investigated. The 23-mer template strand 5'-d(AAC G*C-1 T-2 ACC ATC CGA ATT CGC CC), I (dG* = (+)-trans- and (-)-trans-anti-BPDE-N2-dG), was annealed with primer strands 18, 19, or 20 bases long. Complex formation of these template-primer strands with KF- (exonuclease-free) at different enzyme concentrations was determined using polyacrylamide gel mobility shift assays in the absence of dNTPs. The lesion dG* causes an increase in the dissociation constants, K(d), of the monomeric, 1:1 KF-/DNA template-primer complexes by factors of 10-15 when the 3'-end base of the primer strand is positioned either opposite dG*, or opposite dC-1 in I, and the shapes of the binding isotherms are sigmoidal. The sigmoidal shapes are attributed to the formation of dimeric 2:1 KF6-/DNA template-primer complexes. In contrast, when the 3'-end of the primer strand extends only to dT-2 in I, the Kd of 1:1 complexes is increased by factors of only 2-3, the shapes of the binding isotherms are hyperbolic and nonsigmoidal and are similar to those observed with the unmodified control, and monomeric KF-/DNA complexes are dominant. The impact of bulky lesions on polymerase/DNA complex formation in polymerase-catalyzed primer extension reactions needs to be taken into account in interpreting the site-specific Michaelis-Menten kinetics of these reactions.

Original languageEnglish (US)
Pages (from-to)11834-11843
Number of pages10
Issue number36
StatePublished - Sep 7 1999


ASJC Scopus subject areas

  • Biochemistry

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