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, 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 (1'R, 3'R, 4'R)- 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 hydrogen-bonding 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.