PMID:10818095
| Citation |
Tuske, S, Singh, K, Kaushik, N and Modak, MJ (2000) The J-helix of Escherichia coli DNA polymerase I (Klenow fragment) regulates polymerase and 3'- 5'-exonuclease functions. J. Biol. Chem. 275:23759-68 |
|---|---|
| Abstract |
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. |
| Links |
PubMed Online version:10.1074/jbc.M001804200 |
| Keywords |
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 |
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