The DnaK-DnaJ-GrpE chaperone system activates inert wild type pi initiator protein of R6K into a form active in replication initiation.
The plasmid R6K is an interesting model system for investigating initiation of DNA replication, not only near the primary binding sites of the initiator protein pi but also at a distance, caused by pi -mediated DNA looping. An important milestone in the mechanistic analysis of this replicon was the development of a reconstituted replication system consisting of 22 different highly purified proteins (Abhyankar, M. A., Zzaman, S., and Bastia, D. (2003) J. Biol. Chem. 278, 45476-45484). Although the in vitro reconstituted system promotes ori gamma-specific initiation of replication by a mutant form of the initiator called pi*, the wild type (WT) pi is functionally inert in this system. Here we show that the chaperone DnaK along with its co-chaperone DnaJ and the nucleotide exchange factor GrpE were needed to activate WT pi and caused it to initiate replication in vitro at the correct origin. We show further that the reaction was relatively chaperone-specific and that other chaperones, such as ClpB and ClpX, were incapable of activating WT pi. The molecular mechanism of activation appeared to be a chaperone-catalyzed facilitation of dimeric inert WT pi into iteron-bound monomers. Protein-protein interaction analysis by enzyme-linked immunosorbent assay revealed that, in the absence of ATP, DnaJ directly interacted with pi but its binary interactions with DnaK and GrpE and with ClpB and ClpX were at background levels, suggesting that pi is recruited by protein-protein interaction with DnaJ and then fed into the DnaK chaperone machine to promote initiator activation.
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Shamsu
S
Zzaman
Jagan M
JM
Reddy
Deepak
D
Bastia
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001665:Binding Sites; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000502:physiology*; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000502:physiology*; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism*; D011485:Protein Binding; D015534:Trans-Activators / Q000378:metabolism*
D017931:DNA Primers; D004268:DNA-Binding Proteins; C494635:DnaJ protein, E coli; D029968:Escherichia coli Proteins; C489400:GrpE protein, E coli; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D015534:Trans-Activators; C045264:replication initiator protein; D000255:Adenosine Triphosphate; D004247:DNA; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli; C069775:ClpB protein, E coli; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases