Activation of DNA Binding by the Monomeric Form of the P1 Replication Initiator RepA by Heat Shock Proteins DnaJ and DnaK

Abstract

RepA protein of plasmid P1 binds to arrays of 19 bp repeat sequences (iterons) and mediates initiation of replication and its control. Escherichia coli heat shock proteins DnaJ and DnaK can stimulate iteron binding activity of RepA in an ATP-dependent fashion. It has been proposed that RepA binds to DNA as monomers and that the stimulation in binding involves monomerization of RepA dimers which are inactive in the binding reaction. RepA-iteron and RepA-RepA interactions have been measured in this study to determine the equilibrium constants of the two reactions. The apparent K D value for RepA-iteron binding decreased from 10 nM to no more than 0·2 nM at increasing concentrations of the heat shock proteins. The stimulation of binding appears to be due to an increase in active RepA fraction and not to a change in the maximum binding capacity of the active species. This view was deduced from measurements of active RepA fraction, which increased in the presence of heat shock proteins, and from measurements of dissociation rate constants, which were independent of the heat shock protein concentrations. Accounting for the active fractions, the true K D value was estimated to be 0·10(±0·09) nM in 20 mM Tris·HCl (pH 8), 100 mM NaCl, 40 mM KCl, 10 mM MgCl 2, 1 mM dithiothreitol, 0·1 mM EDTA, ATP (50 μM), bovine serum albumin (50 μg/ml), calf thymus DNA (50 μg/ml) and glycerol (5%). The dissociation rate constant was 1·5 × 10 -2 s -1 and the calculated association rate constant was 1·5 × 10 8 M -1 s -1. Ultracentrifugation analyses of RepA at 15,000 r.p.m. in the above buffer but without ATP, bovine serum albumin, calf thymus DNA and glycerol, revealed that the protein was in monomer-direct equilibrium with a K D of 2·6(±0.2) μM at 5°C. Therefore, at protein concentrations used in the binding reactions, RepA is monomeric (>99·5%), in confirmation of the earlier result that RepA binds as a monomer. It follows that the species that is stimulated to bind by the heat shock proteins is also a monomeric form of RepA.