Primer length dependence of binding of DNA polymerase I Klenow fragment to template-primer complexes containing site-specific bulky lesions.
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 N(2) 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 GC-(1) T(-)(2) ACC ATC CGA ATT CGC CC), I (dG = (+)-trans- and (-)-trans-anti-BPDE-N(2)-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 KF(-)/DNA template-primer complexes. In contrast, when the 3'-end of the primer strand extends only to dT(-)(2) in I, the K(d) 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.
Rechkoblit
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Chemistry Department, New York University, New York, New York 10003-5180, USA.
Amin
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Geacintov
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D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry; D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism*; D019281:Dimerization; D011485:Protein Binding; D013379:Substrate Specificity; D013698:Templates, Genetic
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; D004247:DNA; D004256:DNA Polymerase I