The hydrogen bonding interactions between the Klenow fragment of Escherichia coli DNA polymerase I with the proofreading exonuclease inactivated (KF(-)) and the minor groove of DNA were examined with modified oligodeoxynucleotides in which 3-deazaguanine (3DG) replaced guanine. This substitution would prevent a hydrogen bond from forming between the polymerase and that one site on the DNA. If the hydrogen bonding interaction were important, then we should observe a decrease in the rate of reaction. The steady-state and pre-steady-state kinetics of DNA replication were measured with 10 different oligodeoxynucleotide duplexes in which 3DG was placed at different positions. The largest decrease in the rate of replication was observed when 3DG replaced guanine at the 3'-terminus of the primer. The effect of this substitution on mispair extension and formation was then probed. The G to 3DG substitution at the primer terminus decreased the k(pol) for the extension past G/C, G/A, and G/G base pairs but not the G/T base pair. The G to 3DG substitution at the primer terminus also decreased the formation of correct base pairs as well as incorrect base pairs. However, in all but two mispairs, the effect on correct base pairs was much greater than that of mispairs. These results indicate that the hydrogen bond between Arg668 and the minor groove of the primer terminus is important in the fidelity of both formation and extension of mispairs. These experiments support a mechanism in which Arg668 forms a hydrogen bonding fork between the minor groove of the primer terminus and the ring oxygen of the deoxyribose moiety of the incoming dNTP to align the 3'-hydroxyl group with the alpha-phosphate of the dNTP. This is one mechanism by which the polymerase can use the geometry of the base pairs to modulate the rate of formation and extension of mispairs.

Interactions between the minor groove of the DNA and DNA polymerases appear to play a major role in the catalysis and fidelity of DNA replication. In particular, Arg 668 of Escherichia coli DNA polymerase I (Klenow fragment) makes a critical contact with the N-3-position of guanine at the primer terminus. We investigated the interaction between Arg 668 and the ring oxygen of the incoming deoxynucleotide triphosphate (dNTP) using a combination of site-specific mutagenesis of the protein and atomic substitution of the DNA and dNTP. Hydrogen bonds from Arg 668 were probed with the site-specific mutant R668A. Hydrogen bonds from the DNA were probed with oligodeoxynucleotides containing either guanine or 3-deazaguanine (3DG) at the primer terminus. Hydrogen bonds from the incoming dNTP were probed with (1R,3R,4R)-1-[3-hydroxy-4-(triphosphorylmethyl)cyclopent-1-yl]uracil (dcUTP), an analog of dUTP in which the ring oxygen of the deoxyribose moiety was replaced by a methylene group. We found that the pre-steady-state parameter k pol was decreased 1,600 to 2,000-fold with each of the single substitutions. When the substitutions were combined, there was no additional decrease (R668A and 3DG), a 5-fold decrease (3DG and dcUTP), and a 50-fold decrease (R668A and dcUTP) in k pol . These results are consistent with a hydrogenbonding fork from Arg 668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication.The high fidelity of DNA replication synthesis is accomplished despite the similarity in energy between correctly and incorrectly paired bases (1, 2). Since inter-strand hydrogen bonds cannot account for this selectivity (3, 4), other mechanisms have been proposed to supply the fidelity (reviewed in Ref. 5). These include solvation (6), base stacking (7, 8), steric exclusion (3, 4, 9), and minor groove binding (10). These mechanisms are not mutually exclusive and may all enhance selection for the correct base pairs.The minor groove has been suggested to be a site by which polymerases can check geometry because the O 2 -position of pyrimidines and the N-3-position of purines occupy similar spatial orientations as well as being hydrogen bond acceptors (10). Crystal structures of DNA polymerases bound to DNA have shown that there are many interactions between the protein and the minor groove of DNA (11-15). Site-directed mutagenesis studies have implicated several amino acid residues as being important for catalysis and fidelity of DNA replication (15)(16)(17)(18)(19)(20). Similarly, the use of purine analogs such as 3-deazaadenine (21, 22), 3-deazaguanine (3DG) 1 (23, 24), 9-methyl-1H-imidazo[4,5-b]pyridine and 4-methylbenzimidazole (25,26), and the pyrimidine analogs difluorotoluene (25), 2-aminopyridine, and 3-methyl-2-pyridone (27) also have implicated the minor groove of the DNA as being crucial.X-ray crystallographic studies of polymerases that have a structure similar to that of KF Ϫ (Taq (13) Bacillus stearothermophilus (12) and T7 (14)...

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O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs O(6)-methylguanine (O(6)mG) by transferring the methyl group from the DNA to a cysteine residue on the protein. The kinetics of this reaction was examined by reacting an excess of AGT (0-300 nM) with [5'-(32)P]-labeled oligodeoxynucleotides (0.5 nM) of the sequence 5'-CGT GGC GCT YZA GGC GTG AGC-3' in which Y or Z was G or O(6)mG, annealed to its complementary strand. The reactions, conducted at 25 degrees C, were quenched by the addition of 0.1 N NaOH at various times, and the extents of reaction were monitored by ion exchange HPLC with radiochemical detection. The time courses followed first-order kinetics. The first-order rate constants were plotted against the initial concentration of AGT and fitted to the hyperbolic equation k(obs) = k(inact)[AGT](0)/(K(S) + [AGT](0)). The K(S) values for hAGT of 81-91 nM are 10-fold lower than the dissociation constants of hAGT (C145S) to unmodified and O(6)mG-containing DNA obtained by EMSA and indicate that AGT has a preference for binding to O(6)mG in DNA. The proteins reacted with DNA in which Y = O(6)mG and Z = G faster than Y = G and Z = O(6)mG due to an approximately 10-fold increase in k(inact). These results suggest that the sequence specificity in the repair of O(6)mG is manifested in the methyl transfer not in the O(6)mG recognition step.

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