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

• Published in: The Journal of biological chemistry 2004-08, Vol.279 (32), p.33043-33046
• Main Authors: Meyer, Aviva S., Blandino, Maureen, Spratt, Thomas E.
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.C400232200

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.