The J-helix of Escherichia coli DNA polymerase I (Klenow fragment) regulates polymerase and 3'- 5'-exonuclease functions

J Biol Chem. 2000 Aug 4;275(31):23759-68. doi: 10.1074/jbc.M001804200.


To assess the functional importance of the J-helix region of Escherichia coli DNA polymerase I, we performed site-directed mutagenesis of the following five residues: Asn-675, Gln-677, Asn-678, Ile-679, and Pro-680. Of these, the Q677A mutant is polymerase-defective with no change in its exonuclease activity. In contrast, the N678A mutant has unchanged polymerase activity but shows increased mismatch-directed exonuclease activity. Interestingly, mutation of Pro-680 has a Q677A-like effect on polymerase activity and an N678A-like effect on the exonuclease activity. Mutation of Pro-680 to Gly or Gln results in a 10-30-fold reduction in k(cat) on homo- and heteropolymeric template-primers, with no significant change in relative DNA binding affinity or K(m)((dNTP)). The mutants P680G and P680Q also showed a nearly complete loss in the processive mode of DNA synthesis. Since the side chain of proline is generally non-reactive, mutation of Pro-680 may be expected to alter the physical form of the J-helix itself. The biochemical properties of P680G/P680Q together with the structural observation that J-helix assumes helical or coiled secondary structure in the polymerase or exonuclease mode-bound DNA complexes suggest that the structural alteration in the J-helix region may be responsible for the controlled shuttling of DNA between the polymerase and the exonuclease sites.

Publication types

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

MeSH terms

  • Base Pair Mismatch
  • Catalytic Domain*
  • DNA Polymerase I / genetics
  • DNA Polymerase I / metabolism*
  • DNA, Single-Stranded / metabolism
  • DNA-Binding Proteins / metabolism
  • Escherichia coli
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism*
  • Kinetics
  • Mutagenesis, Site-Directed
  • Nucleic Acid Conformation
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism*
  • Poly dA-dT / metabolism


  • DNA, Single-Stranded
  • DNA-Binding Proteins
  • Peptide Fragments
  • Poly dA-dT
  • DNA Polymerase I
  • Exodeoxyribonucleases
  • Exodeoxyribonuclease V