In vitro roles of Escherichia coli DnaJ and DnaK heat shock proteins in the replication of oriC plasmids.
Heat shock proteins have been shown to be involved in many cellular processes in procaryotic and eucaryotic cells. Using an in vitro DNA replication assay, we show that DNA synthesis initiated at the chromosomal origin of replication of Escherichia coli (oriC) is considerably reduced in enzyme extracts isolated from cells bearing mutations in the dnaK and dnaJ genes, which code for heat shock proteins. Furthermore, unlike DNA synthesis in wild-type extracts, residual DNA synthesis in dnaK and dnaJ extracts is thermosensitive. Although thermosensitivity can be complemented by the addition of DnaK and DnaJ proteins, restoration of near wild-type replication levels requires supplementary quantities of purified DnaA protein. This key DNA synthesis initiator protein is shown to be adsorbed to DnaK affinity columns. These results suggest that at least one of the heat shock proteins. DnaK, exerts an effect on the initiation of DNA synthesis at the level of DnaA protein activity. However, our observation of normal oriC plasmid transformation ratios and concentrations in heat shock mutants at permissive temperatures would suggest that heat shock proteins play a role in DNA replication mainly at high temperatures or under other stressful growth conditions.
Malki
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A
Institute Jacques Monod, Centre National de la Recherche Scientifique, Université Paris, France.
Hughes
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P
Kohiyama
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D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002846:Chromatography, Affinity; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D010957:Plasmids; D013696:Temperature; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C063842:dnaK protein, E coli