ABSTRACT
Control of DNA replication initiation is essential for cell growth. A unifying characteristic of DNA replication initiator proteins is their distinctive AAA+ nucleotide-binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains to form an active initiation complex. Recently we developed a unique cross-linking assay that specifically detects ATP-dependent DnaA helix assembly. Here we have utilized this assay to show that two DnaA regulatory proteins in Bacillus subtilis, YabA and DnaD, inhibit DnaA helix formation. These results, in combination with our previous finding that the regulatory factor Soj/ParA also targets DnaA filament formation, highlight the critical importance of regulating DnaA helix formation during the initiation reaction. Moreover, these observations lead us to suggest that DnaA oligomerization may be the main regulatory step of the initiator assembly pathway in B. subtilis, in contrast to the prevailing model of bacterial DNA replication based on Escherichia coliâ DnaA where ATP binding appears to be the targeted activity.
Subject(s)
Bacillus subtilis/physiology , Bacterial Proteins/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , Protein Multimerization , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Models, MolecularABSTRACT
When starved, Bacillus subtilis cells can enter the developmental programme of endospore formation by activation of the master transcriptional regulator Spo0A. Correct chromosome copy number is crucial for the production of mature and fully resistant spores. The production and maintenance of one chromosome for the mother cell and one copy for the forespore requires accurate co-ordination between DNA replication and initiation of sporulation. Here, we show that Spo0A regulates chromosome copy number by directly binding to a number of Spo0A binding sites that are present near the origin of replication (oriC). We demonstrate that cells lacking three specific Spo0A binding sites at oriC display increased chromosome copy numbers when sporulation is induced. Our data support the hypothesis that Spo0A directly controls DNA replication during sporulation by binding to oriC.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/metabolism , Chromosomes, Bacterial/genetics , Gene Dosage , Gene Expression Regulation, Developmental , Replication Origin , Spores, Bacterial/growth & development , Transcription Factors/metabolism , Bacillus subtilis/growth & development , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Chromosomes, Bacterial/metabolism , Gene Expression Regulation, Bacterial , Protein Binding , Spores, Bacterial/genetics , Spores, Bacterial/metabolism , Transcription Factors/geneticsABSTRACT
Control of DNA replication initiation is essential for normal cell growth. A unifying characteristic of DNA replication initiator proteins across the kingdoms of life is their distinctive AAA+ nucleotide-binding domains. The bacterial initiator DnaA assembles into a right-handed helical oligomer built upon interactions between neighbouring AAA+ domains, that in vitro stretches DNA to promote replication origin opening. The Bacillus subtilis protein Soj/ParA has previously been shown to regulate DnaA-dependent DNA replication initiation; however, the mechanism underlying this control was unknown. Here, we report that Soj directly interacts with the AAA+ domain of DnaA and specifically regulates DnaA helix assembly. We also provide critical biochemical evidence indicating that DnaA assembles into a helical oligomer in vivo and that the frequency of replication initiation correlates with the extent of DnaA oligomer formation. This work defines a significant new regulatory mechanism for the control of DNA replication initiation in bacteria.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Replication/physiology , DNA, Bacterial/biosynthesis , DNA-Binding Proteins/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , DNA, Bacterial/genetics , DNA-Binding Proteins/genetics , Hydrolysis , Models, Molecular , Molecular Sequence Data , Point Mutation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Replication OriginABSTRACT
Control of DNA replication initiation is essential for bacterial cells to co-ordinate the faithful replication and segregation of their genetic material. The Bacillus subtilis ATPase Soj is a dynamic protein that regulates DNA replication initiation by either inhibiting or activating the DNA replication initiator protein DnaA. Here we report that the key event which switches Soj regulatory activity is a transition in its oligomeric state from a monomer to an ATP-dependent homodimer capable of DNA binding. We show that the DNA binding activity of the Soj dimer is required both for activation of DNA replication initiation and for interaction with Spo0J. Finally, we demonstrate that Spo0J inhibits Soj dimerization by stimulating Soj ATPase activity. The data provide a molecular explanation for the dichotomous regulatory activities of Soj, as well as assigning unique Soj conformations to distinct cellular localization patterns. We discuss how the regulation of Soj ATPase activity by Spo0J could be utilized to control the initiation of DNA replication during the cell cycle.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , Adenosine Triphosphatases/metabolism , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , DNA-Binding Proteins/genetics , Gene Expression Regulation, Bacterial , Mutation , Protein Multimerization , Spores, Bacterial/genetics , Spores, Bacterial/metabolismABSTRACT
Mutations in DNA replication initiator genes in both prokaryotes and eukaryotes lead to a pleiotropic array of phenotypes, including defects in chromosome segregation, cytokinesis, cell cycle regulation and gene expression. For years, it was not clear whether these diverse effects were indirect consequences of perturbed DNA replication, or whether they indicated that DNA replication initiator proteins had roles beyond their activity in initiating DNA synthesis. Recent work from a range of organisms has demonstrated that DNA replication initiator proteins play direct roles in many cellular processes, often functioning to coordinate the initiation of DNA replication with essential cell-cycle activities. The aim of this review is to highlight these new findings, focusing on the pathways and mechanisms utilized by DNA replication initiator proteins to carry out a diverse array of cellular functions.
