PMID:15210707
| Citation |
Meyer, AS, Blandino, M and Spratt, TE (2004) 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. J. Biol. Chem. 279:33043-6 |
|---|---|
| Abstract |
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. Hydrogen bonds from Arg668 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 kpol 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 kpol. These results are consistent with a hydrogen-bonding fork from Arg668 to the primer terminus and incoming dNTP. These interactions may play an important role in fidelity as well as catalysis of DNA replication. |
| Links |
PubMed Online version:10.1074/jbc.C400232200 |
| Keywords |
Arginine/metabolism; Binding Sites; Catalysis; DNA Polymerase I/chemistry; DNA Polymerase I/genetics; DNA Polymerase I/metabolism; DNA Replication; Escherichia coli/enzymology; Guanine/analogs & derivatives; Guanine/metabolism; Hydrogen Bonding; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides/metabolism; Uridine Triphosphate/analogs & derivatives |
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