Defect in general priming conferred by linker region mutants of Escherichia coli dnaB.
The dnaB gene of Escherichia coli encodes a bifunctional primase accessory protein/helicase necessary for chromosomal replication. Monomers of DnaB comprise two trypsin-resistant domains connected by a 45-amino-acid linker. To investigate the role of the linker in the structure and function of DnaB, we have purified and characterized three DnaB mutant proteins having single amino acid substitutions in the linker. We find that the mutant proteins retain the two-domain structure and assemble into hexamers that may be less stable than hexamers formed by wild-type DnaB. These mutant hexamers have hydrodynamic properties slightly different from those of the wild type, suggestive of a more open structure. The mutant proteins had reduced or absent ability to stimulate primase and also exhibited slight alterations in ATPase activity compared with the wild type. We conclude that the linker region promotes primase-DnaB interaction, but this effect may be indirect. We propose a model involving repositioning of N-terminal domains to explain the properties of the mutant proteins.
Stordal
L
L
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960, USA.
Maurer
R
R
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019915:DNA Primase; D004261:DNA Replication; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D015394:Molecular Structure; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D009154:Mutation; D012316:RNA Nucleotidyltransferases / Q000378:metabolism
D001426:Bacterial Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; D053488:DnaB Helicases