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

Abstract

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.

The J-helix of Escherichia coli DNA Polymerase I (Klenow Fragment) Regulates Polymerase and 3′– 5′-Exonuclease Functions

“…Table II gives relative DNA binding affinity values for WT and various J-helix mutant derivatives. The relative DNA binding affinity obtained by our cross-linking experiments is 12 nM for WT, which is in good agreement with the values determined by pre-steady state kinetic methods and gel mobility shift assays (18,19). Only slight variation in the relative DNA binding affinity (3-fold increase or decrease) was observed for different J-helix mutant enzymes, indicating little or no effect of these substitutions on the template-primer binding affinity.…”

trans-Lesion Synthesis Past Bulky Benzo[a]pyrene Diol Epoxide N2-dG and N6-dA Lesions Catalyzed by DNA Bypass Polymerases

“…sequence is the codon 61 mutation hotspot of the N-ras gene. The carcinogen-containing dG*-modified 33-mers and dA*-modified 45-mers were constructed by ligating the appropriate combinations of appropriate oligonucleotides by T4 DNA ligase and purified as described previously (28,56 32 P]ATP. The unmodified and modified DNA template-primer complexes were prepared by annealing the template and primer strands (15% excess of the template strand) and by heating the solution to 90°C, followed by slow cooling to room temperature.…”

The Spacious Active Site of a Y-Family DNA Polymerase Facilitates Promiscuous Nucleotide Incorporation Opposite a Bulky Carcinogen-DNA Adduct

“…This oligodeoxynucleotide sequence was designed to prevent slippage-mediated bypass of the adduct (45). The 42-mers containing a single (ϩ)-ta[BP]G were constructed by ligation methods (45,65,68); the modified 11-mers and flanking 12-and 19-mers were annealed with a complementary 38-mer template strand, and then the three oligonucleotides were ligated to one another using T4 DNA ligase (New England Biolabs, Inc., Beverly, MA) as described previously (47,68,71). The intact 42-mers were purified using 20% PAGE with 8 M urea, visualized using ethidium bromide and then excised from the gel.…”