FUNCTION OF THE GRPE HEAT-SHOCK PROTEIN IN BIDIRECTIONAL UNWINDING AND REPLICATION FROM THE ORIGIN OF PHAGE-GAMMA

The initiation of DNA replication by phage lambda depends on a special ized nucleoprotein structure that provides for the precise localizatio n and activity of the Escherichia coli DnaB helicase at the lambda rep lication origin. Previous work has shown that the DnaJ and DnaK heat s hock 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 unidirectiona l DNA unwinding and replication, whereas replication in vivo is mainly bidirectional. Based on recent replication work indicating an importa nt role for the GrpE heat shock protein, we have used electron microsc opy to study the action of GrpE in the DNA unwinding and replication r eactions. We have found that GrpE acts with DnaJ and DnaK to facilitat e the unwinding reaction at low concentrations of DnaK. In the presenc e 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 sugge st that reactions including GrpE result in more complete disassembly o f the preinitiation nucleoprotein structure, thus allowing replication to proceed in both directions from the origin.