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

To assess the functional importance of the J-helix region of Escherichia co li DNA polymerase I, we performed site-directed mutagenesis of the followin g 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 N 678A-like effect on the exonuclease activity. Mutation of Pro-680 to Gly or Gin results in a 10-30-fold reduction in k(cat) on homo- and heteropolymer ic template-primers, with no significant change in relative DNA binding aff inity or K-m(dNTP). The mutants P680G and P680Q also showed a nearly comple te loss in the processive mode of DNA synthesis. Since the side chain of pr oline is generally non-reactive, mutation of Pro-680 may be expected to alt er the physical form of the J-helix itself. The biochemical properties of P 680G/P680Q together with the structural observation that J-helix assumes he lical or coiled secondary structure in the polymerase or exonuclease mode-b ound DNA complexes suggest that the structural alteration in the J-helix re gion may be responsible for the controlled shuttling of DNA between the pol ymerase and the exonuclease sites.