The J-helix of Escherichia coli DNA Polymerase I (Klenow Fragment) Regulates Polymerase and 3′– 5′-Exonuclease Functions

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 homoand 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.The first solved crystal structure of the Klenow fragment (KF) 1 (1) of Escherichia coli DNA polymerase I revealed an enzyme modularly constructed into two domains as follows: (i) an ϳ200-residue N-terminal domain and (ii) an ϳ400-residue C-terminal domain. The C-terminal domain contained an ϳ35-Å deep cleft and a dNMP-bound N-terminal domain. Model building of B-DNA within the C-terminus led the authors (1) to propose this region as the polymerase-active site and the N-terminal domain as the 3Ј-exonuclease site. Subsequent attempts to solve the co-crystal structure of DNA complexed with KF resulted in identification of an editing complex with single-stranded DNA bound at the previously proposed exonuclease domain (2, 3). The enzyme-nucleic acid interactions observed in this complex and the subsequent structural and biochemical studies (2-5) provided a rational mechanism for the hydrolysis of dNMP from the 3Ј terminus and identified key amino acid residues essential for the exonuclease activity.Recent resolution of T7 and KlenTaq DNA polymerase (6 -8) crystal structure complexes has provided detailed information on the interactions between DNA-dNTP and protein in binary and ternary complexes. In addition, these crystal structures have revealed conformational changes in various enzyme subdomains and DNA that appear to be a prerequisite for the formation of proper active site geometry for dNTP incorporation. A detailed examination of the binary and ternary complex crystal structures of the pol I family of DNA polymerases has revealed sequential conformational changes, relative to the unliganded protein, upon template-primer and dNTP binding, respectively (7-...

Presence of 18-Å Long Hydrogen Bond Track in the Active Site of Escherichia coli DNA Polymerase I (Klenow Fragment)

“…A change in the position of Gln 677 would result in the destabilization of the H-bonding track arrangement, which in turn would not permit the binding of enzyme to template-primer. Whereas the polymerase activity of the P680G mutant enzyme was severely reduced, the mutant enzyme was extremely efficient in the 3Ј-5Ј proofreading activity (35). We have noted a similar increase in the efficiency of 3Ј-5Ј exonuclease activity with the Q677A mutant species.…”

“…Our activity and steady-state kinetic data also show severe loss in the polymerase activity of both nonhomologous Q677A and homologous Q677N mutant enzymes (Table II). In fact, the polymerase activity of the P680G mutant of KF was also reduced to the same extent as that seen for Gln 677 mutant enzymes (35). Therefore, it is possible that the reduced activity of P680G was a reflection of the activity of the enzyme because of the altered position of Gln 677 .…”

“…This in turn allows Gln 677 to make contacts with His 881 and Gln 879 . The mutant Q677A enzyme is an inactive enzyme (35,36). Our activity and steady-state kinetic data also show severe loss in the polymerase activity of both nonhomologous Q677A and homologous Q677N mutant enzymes (Table II).…”

“…However, the helical structure is maintained when the binding of the template-primer occurs in the 3Ј-5Ј exonuclease mode (9). We have previously shown that helix to coil transition (or vice versa) of the J-helix plays a crucial role in the switching of the template-primer from the polymerase to the exonuclease site, as judged from the properties of P680G (35). Upon the binding of template-primer to the enzyme in the polymerase mode, repositioning of the side chain of Gln 677 has also been noted.…”

Mutations in the Spacer Region of Drosophila Mitochondrial DNA Polymerase Affect DNA Binding, Processivity, and the Balance between Pol and Exo Function

“…Both biochemical and structural studies of family A DNA polymerases support the possibility that amino acid residues required for enzyme function lie outside the catalytic active site regions (19,(33)(34)(35)(36). Because structural information on pol ␥-␣ is very limited, a secondary structure prediction algorithm (PredictProtein) was used to identify the positions of potential ␣-helices and ␤-strands in the spacer region, and this information was taken into consideration in the generation of triple and nested single alanine mutations that were introduced as substitutions for highly conserved residues in the ␥1, ␥3, and ␥4 elements (Fig.…”