Characterization of the Unique C Terminus of theEscherichia coli τ DnaX Protein: MONOMERIC C-τ BINDS α AND DnaB AND CAN PARTIALLY REPLACE τ IN RECONSTITUTED REPLICATION FORKS

H.Garry Dallmann,Sungsub Kim,Arthur E Pritchard,Kenneth J Marians,Charles S Mchenry

doi:10.1074/jbc.m909257199

Abstract

A contact between the dimeric tau subunit within the DNA polymerase III holoenzyme and the DnaB helicase is required for replication fork propagation at physiologically-relevant rates (Kim, S., Dallmann, H. G., McHenry, C. S., and Marians, K. J. (1996) Cell 84, 643-650). In this report, we exploit the OmpT protease to generate C-tau, a protein containing only the unique C-terminal sequences of tau, free of the sequences shared with the alternative gamma frameshifting product of dnaX. We have established that C-tau is a monomer by sedimentation equilibrium and sedimentation velocity ultracentrifugation. Monomeric C-tau binds the alpha catalytic subunit of DNA polymerase III with a 1:1 stoichiometry. C-tau also binds DnaB, revealed by a coupled immunoblotting method. C-tau restores the rapid replication rate of inefficient forks reconstituted with only the gamma dnaX gene product. The acceleration of the DnaB helicase can be observed in the absence of primase, when only leading-strand replication occurs. This indicates that C-tau, bound only to the leading-strand polymerase, can trigger the conformational change necessary for DnaB to assume the fast, physiologically relevant form.

Highlights

A contact between the dimeric ␶ subunit within the DNA polymerase III holoenzyme and the DnaB helicase is required for replication fork propagation at physiologically-relevant rates
The ␶ subunit of the DNA polymerase III holoenzyme can be cleaved by the OmpT protease between two exposed adjacent lysines to generate a protein two residues shorter than authentic ␥, ␥p, and the intact C terminus unique to ␶, C-␶ [28]
The sedimentation equilibrium data were not consistent with any detectable higher molecular weight forms in equilibrium with the 1:1 complex. This shows that binding of C-␶ does not trigger ␣ to dimerize, at least to a detectable level and in the absence of other components found at the replication fork

Summary

EXPERIMENTAL PROCEDURES

DNAs, Enzymes, and Replication Proteins—Alkaline phosphatase was from Roche Molecular Biochemicals. Isolation of ␤-Template DNA Complexes—A reaction mixture (120 ␮l) containing 0.83 nM TFII DNA, 82 nM ␤, 41 nM ␥-complex, 1.1 mM SSB, 50 mM Hepes-KOH (pH 7.9), 12 mM Mg(OAc) mM DTT, 2 mM ATP, 80 mM KCl, 0.1 mg/ml BSA, 200 mM each of GTP, CTP, and UTP, and 40 mM dCTP and dGTP (all 4 NTPs and the 2 dNTPs are included just so that the pooled material would be in the same reaction buffer as the rolling circle reaction) was incubated for 10 min at 30 °C and filtered through a 5-ml Bio-Gel A-150m (Bio-Rad) column equilibrated with Buffer R. Replication Fork Rates—An aliquot (110 ␮l) of the peak fraction containing the ␤1⁄7TFII DNA complex was incubated in a total reaction

Method

RESULTS

DISCUSSION