Primer Length Dependence of Binding of DNA Polymerase I Klenow Fragment to Template-Primer Complexes Containing Site-Specific Bulky Lesions

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• 作者：Olga Rechkoblit ; Shantu Amin ; and Nicholas E. Geacintov
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：September 7, 1999
• 年：1999
• 卷：38
• 期：36
• 页码：11834 - 11843
• 全文大小：195K
• 年卷期：v.38,no.36(September 7, 1999)
• ISSN：1520-4995

文摘

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 adverselyaffect 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 ofPol I (Klenow fragment, KF)/template-primer complexes has been investigated. The 23-mer templatestrand 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 thesetemplate-primer strands with KF^- (exonuclease-free) at different enzyme concentrations was determinedusing polyacrylamide gel mobility shift assays in the absence of dNTPs. The lesion dG* causes an increasein the dissociation constants, K_d, of the monomeric, 1:1 KF^-/DNA template-primer complexes by factorsof 10-15 when the 3'-end base of the primer strand is positioned either opposite dG*, or opposite dC_-1in I, and the shapes of the binding isotherms are sigmoidal. The sigmoidal shapes are attributed to theformation of dimeric 2:1 KF^-/DNA template-primer complexes. In contrast, when the 3'-end of theprimer strand extends only to dT_-2 in I, the K_d of 1:1 complexes is increased by factors of only 2-3, theshapes of the binding isotherms are hyperbolic and nonsigmoidal and are similar to those observed withthe unmodified control, and monomeric KF^-/DNA complexes are dominant. The impact of bulky lesionson polymerase/DNA complex formation in polymerase-catalyzed primer extension reactions needs to betaken into account in interpreting the site-specific Michaelis-Menten kinetics of these reactions.