Fidelity of mispair formation and mispair extension is dependent on the interaction between the minor groove of the primer terminus and Arg668 of DNA polymerase I of Escherichia coli.

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.

Escherichia coli DNA polymerase I (Klenow fragment) uses a hydrogen-bonding fork from Arg668 to the primer terminus and incoming deoxynucleotide triphosphate to catalyze DNA replication.

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, Arg668 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 Arg668 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.

O6-alkylguanine-DNA alkyltransferases repair O6-methylguanine in DNA with Michaelis-Menten-like kinetics.

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.