Dissociation kinetics of RepA dimers: implications for mechanisms of activation of DNA binding by chaperones.
BACKGROUND
The replication initiator of plasmid P1, RepA, binds DNA as monomer. The binding is stimulated by the chaperones DnaJ, DnaK and GrpE of Escherichia coli. Two models of chaperone action have been proposed. (i) Chaperones dissociate RepA dimers, which are inactive in DNA binding, into active monomers. (ii) The dissociation occurs spontaneously but the monomeric products require the chaperones for refolding into the active form. The latter model was based on the observation that RepA diluted 1000-fold below the K(D) for dimer dissociation, still required the chaperones for DNA binding.


RESULTS
We have confirmed that under the condition of DNA binding experiments, the RepA dimers dissociate reversibly into monomers with a K(D) value of 1.1 +/- 0.1 microM. In the vicinity of this concentration, the sedimentation coefficient of RepA was concentration dependent, allowing estimation of s(20,w) coefficients for the RepA monomer (2.95 S) and dimer (4.01 S). Dynamic light scattering experiments indicated an increase of the monomer fraction within 5 min of RepA dilution. Circular dichroism (CD) measurements were consistent with these results.


CONCLUSIONS
RepA monomerization is efficient without the mediation of chaperones. They are required to activate RepA most likely because they are needed to re-fold RepA monomers.
Chattoraj
D K
DK
Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, MD 20892, USA.
Ghirlando
R
R
Park
K
K
Dibbens
J A
JA
Lewis
M S
MS
D002942:Circular Dichroism; D004265:DNA Helicases; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D008027:Light; D018832:Molecular Chaperones / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D012542:Scattering, Radiation; D015534:Trans-Activators; D014461:Ultracentrifugation
D004268:DNA-Binding Proteins; D018832:Molecular Chaperones; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004265:DNA Helicases