DNA Unwinding by Escherichia coli DNA Helicase I (TraI) Provides Evidence for a Processive Monomeric Molecular Motor

Bartek Sikora,Robert L Eoff,Steven W Matson,Kevin D Raney

doi:10.1074/jbc.m604412200

Abstract

The F plasmid TraI protein (DNA helicase I) plays an essential role in conjugative DNA transfer as both a transesterase and a helicase. Previous work has shown that the 192-kDa TraI protein is a highly processive helicase, catalytically separating >850 bp under steady-state conditions. In this report, we examine the kinetic mechanism describing DNA unwinding of TraI. The kinetic step size of TraI was measured under both single turnover and pre-steady-state conditions. The resulting kinetic step-size estimate was approximately 6-8 bp step(-1). TraI can separate double-stranded DNA at a rate of approximately 1100 bp s(-1), similar to the measured unwinding rate of the RecBCD helicase, and appears to dissociate very slowly from the 3' terminus following translocation and strand-separation events. Analyses of pre-steady-state burst amplitudes indicate that TraI can function as a monomer, similar to the bacteriophage T4 helicase, Dda. However, unlike Dda, TraI is a highly processive monomeric helicase, making it unique among the DNA helicases characterized thus far.

Highlights

Generation of a directional force, transport of nucleic acid from one point in the cell to another, and packaging of viral nucleic acid [5,6,7,8]
DNA helicase I (TraI) is a bifunctional protein encoded by the Escherichia coli F-plasmid [9] that contains three functional domains essential for transfer of bacterial genes during conjugation (10 –12)
The results reported provide evidence that TraI can function as a processive monomeric helicase

Summary

EXPERIMENTAL PROCEDURES

Materials—ATP (disodium salt) and Sephadex (G-25) were obtained from Sigma. HEPES, Na4EDTA, ␤-mercaptoethanol, bovine serum albumin, Mg(OAc), KOAc, SDS, xylene cyanol, bromphenol blue, NaCl, glycerol, and KOH were obtained from Fisher. T4 polynucleotide kinase was purchased from New England Biolabs. [␥-32P]ATP was purchased from New England Nuclear. DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA) were purified by preparative PAGE and stored in 10 mM HEPES (pH 7.5) and 1 mM EDTA. All oligonucleotides were resuspended in 10 mM HEPES/1 mM EDTA, pH

Kinetics of the TraI Mechanism of DNA Unwinding

RESULTS

Global kinetic parameters determined from NLLS

DISCUSSION