DnaAcos hyperinitiates by circumventing regulatory pathways that control the frequency of initiation in Escherichia coli.
Mutants of dnaAcos are inviable at 30 degrees C because DnaAcos hyperinitiates, leading to new replication forks that apparently collide from behind with stalled forks, thereby generating lethal double-strand breaks. By comparison, an elevated level of DnaA also induces extra initiations, but lethality occurs only in strains defective in repairing double-strand breaks. To explore the model that the chromosomal level of DnaAcos, or the increased abundance of DnaA, increases initiation frequency by, escaping or overcoming pathways that control initiation, respectively, we developed a genetic selection and identified seqA, datA, dnaN and hda, which function in pathways that either act at oriC or modulate DnaA activity. To assess each pathway's relative effectiveness, we used genetically inactivated strains, and quantified initiation frequency after elevating the level of DnaA. The results indicate that the hda-dependent pathway has a stronger effect on initiation than pathways involving seqA and datA. Testing the model that DnaAcos overinitiates because it fails to respond to one or more regulatory mechanisms, we show that dnaAcos is unresponsive to hda and dnaN, which encodes the beta clamp, and also datA, a locus proposed to titer excess DnaA. These results explain how DnaAcos hyperinitiates to interfere with viability.
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
Magdalena M
MM
Felczak
Jon M
JM
Kaguni
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015698:Genomic Library; D010957:Plasmids
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins