Mapping protein-protein interactions within a stable complex of DNA primase and DnaB helicase from Bacillus stearothermophilus.
For the first time, we demonstrate directly a stable complex between a bacterial DnaG (primase) and DnaB (helicase). Utilizing fragments of both proteins, we are able to dissect interactions within this complex and provide direct evidence that it is the C-terminal domain of primase that interacts with DnaB. Furthermore, this C-terminal domain is sufficient to induce maximal stimulation of the helicase and ATPase activities of DnaB. However, the region of DnaB that interacts with the C-terminal domain of primase appears to comprise a surface on DnaB that includes regions from both of the previously identified N- and C-terminal domains. Using a combination of biochemical and physical techniques, we show that the helicase-primase complex comprises one DnaB hexamer and either two or three molecules of DnaG. Our results show that in Bacillus stearothermophilus the helicase-primase interaction at the replication fork may not be transient, as was shown to be the case in Escherichia coli. Instead, primase appears to interact with the helicase forming a tighter complex with enhanced ATPase and helicase activities.
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Sir William Dunn School of Pathology, University of Oxford, U.K.
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Soultanas
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Wigley
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D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D053488:DnaB Helicases; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D011487:Protein Conformation; D014357:Trypsin
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D019915:DNA Primase; D014357:Trypsin; D004265:DNA Helicases; D053488:DnaB Helicases