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. 2004 Aug 6;279(32):33043-6. doi: 10.1074/jbc.C400232200. Epub 2004 Jun 20.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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


  • Oligodeoxyribonucleotides
  • Guanine
  • Arginine
  • 3-deazaguanine
  • DNA Polymerase I
  • Uridine Triphosphate