Cryptic single-stranded-DNA binding activities of the phage lambda P and Escherichia coli DnaC replication initiation proteins facilitate the transfer of E. coli DnaB helicase onto DNA.
The bacteriophage lambda P and Escherichia coli DnaC proteins are known to recruit the bacterial DnaB replicative helicase to initiator complexes assembled at the phage and bacterial origins, respectively. These specialized nucleoprotein assemblies facilitate the transfer of one or more molecules of DnaB helicase onto the chromosome; the transferred DnaB, in turn, promotes establishment of a processive replication fork apparatus. To learn more about the mechanism of the DnaB transfer reaction, we investigated the interaction of replication initiation proteins with single-stranded DNA (ssDNA). These studies indicate that both P and DnaC contain a cryptic ssDNA-binding activity that is mobilized when each forms a complex with the DnaB helicase. Concomitantly, the capacity of DnaB to bind to ssDNA, as judged by UV-crosslinking analysis, is suppressed upon formation of a P x DnaB or a DnaB x DnaC complex. This novel switch in ssDNA-binding activity evoked by complex formation suggests that interactions of P or DnaC with ssDNA may precede the transfer of DnaB onto DNA during initiation of DNA replication. Further, we find that the lambda O replication initiator enhances interaction of the P x DnaB complex with ssDNA. Partial disassembly of a ssDNA:O x P x DnaB complex by the DnaK/DnaJ/GrpE molecular chaperone system results in the transfer in cis of DnaB to the ssDNA template. On the basis of these findings, we present a general model for the transfer of DnaB onto ssDNA or onto chromosomal origins by replication initiation proteins.
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B A
BA
Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205, USA.
Um
S J
SJ
Huang
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L
McMacken
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R
D001426:Bacterial Proteins / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics*; D003432:Cross-Linking Reagents; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D008957:Models, Genetic; D011485:Protein Binding; D014764:Viral Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; C024013:DNA replication complex protein, Bacteriophage lambda; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins; D014764:Viral Proteins; D004265:DNA Helicases; D053488:DnaB Helicases