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.
Highlights
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
In the crystal structures of pol I family DNA polymerases, the J-helix region has been seen to interact with minor groove and primer phosphate backbone of the template-primer and appears to assume variable conformation depending upon the nature of the enzyme-DNA complex
Gln-677 does not interact with the template-primer in the crystal structure, yet its alanine mutant (Q677A) was polymerase-deficient
Summary
Materials—Mutagen M13 in vitro mutagenesis kit was purchased from Bio-Rad. Sequenase and DNA sequencing reagents were purchased from United States Biochemical Corp. Single dNTP Turnover by WT and Mutant Klenow Fragment—An assay designed to limit observations to a single dNTP incorporation for a KF1⁄7TP complex was carried out as follows: 5 nM 5Ј-32P-labeled 16-mer oligomer, annealed to 46-mer template, was incubated at 23 °C for 5 min with 50 nM WT or mutant KF in a buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM DTT, and 100 M dTTP, in a final volume of 24 l. Time Course of TTP Incorporation on Poly(dA)1⁄7(dT) TemplatePrimer and Determination of Processivity—In order to investigate the rate-limiting step on homopolymeric poly(dA)1⁄7(dT) template-primer by the mutant enzymes, we carried out a time course of dTTP incorporation In this experiment, the extension of the 5Ј-32P-labeled (dT) annealed to poly(dA) template was monitored by gel analysis of the products. The extent of primer remaining at 17-mer position was plotted as a function of time, and the rate constants were calculated directly from the slope of the line
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
