Function of the GrpE heat shock protein in bidirectional unwinding and replication from the origin of phage lambda.
The initiation of DNA replication by phage lambda depends on a specialized nucleoprotein structure that provides for the precise localization and activity of the Escherichia coli DnaB helicase at the lambda replication origin. Previous work has shown that the DnaJ and DnaK heat shock proteins function in the initiation pathway by releasing the DnaB helicase from the initiation complex to carry out localized unwinding of origin DNA. This DnaJ.DnaK pathway results in mainly unidirectional DNA unwinding and replication, whereas replication in vivo is mainly bidirectional. Based on recent replication work indicating an important role for the GrpE heat shock protein, we have used electron microscopy to study the action of GrpE in the DNA unwinding and replication reactions. We have found that GrpE acts with DnaJ and DnaK to facilitate the unwinding reaction at low concentrations of DnaK. In the presence of GrpE, bidirectional unwinding occurs in approximately half of the unwound DNA molecules. In addition, GrpE significantly increases the frequency of replication proceeding leftward from the origin. We suggest that reactions including GrpE result in more complete disassembly of the preinitiation nucleoprotein structure, thus allowing replication to proceed in both directions from the origin.
Wyman
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Division of Biochemistry and Molecular Biology, University of California, Berkeley 94720.
Vasilikiotis
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Ang
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Georgopoulos
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Echols
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D001426:Bacterial Proteins / Q000502:physiology*; D010582:Bacteriophage lambda / Q000235:genetics*; D004265:DNA Helicases / Q000502:physiology*; D004261:DNA Replication; D004274:DNA, Recombinant / Q000096:biosynthesis / Q000737:chemistry / Q000648:ultrastructure; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D006360:Heat-Shock Proteins / Q000502:physiology*; D008854:Microscopy, Electron
D001426:Bacterial Proteins; D004274:DNA, Recombinant; D004279:DNA, Viral; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D006360:Heat-Shock Proteins; D004265:DNA Helicases