Functional dissection of YabA, a negative regulator of DNA replication initiation in
            <i>Bacillus subtilis</i>

Noirot‐Gros, Marie‐Françoise; Velten, Marion; Yoshimura, Mika; McGovern, Stephen; Morimoto, Takahiro; Ehrlich, S. Dusko; Ogasawara, Nagahisa; Polard, Patrice; Naveilhan, Philippe

doi:10.1073/pnas.0506914103

Cited by 77 publications

(155 citation statements)

References 36 publications

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“…Its modular architecture allows for interactions with different binding partners during the various steps of the formation of the active replication forks and coupling to other cellular events (3,15,17,20,22,29,30). In E. coli, DnaA III interactions with itself, Hda, or DnaC are important for the spatial positioning of the ATP-DnaA molecules and their recycling (13,17,31,32).…”

Section: Discussionmentioning

confidence: 99%

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The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

Natrajan¹,

Noirot‐Gros

Zawilak-Pawlik

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial DNA replication requires DnaA, an AAA؉ ATPase that initiates replication at a specific chromosome region, oriC, and is regulated by species-specific regulators that directly bind DnaA. HobA is a DnaA binding protein, recently identified as an essential regulator of DNA replication in Helicobacter pylori. We report the crystal structure of HobA in complex with domains I and II of DnaA (DnaA I-II ) from H. pylori, the first structure of DnaA bound to one of its regulators. Biochemical characterization of the complex formed shows that a tetramer of HobA binds four DnaA I-II molecules, and that DnaA I-II is unable to oligomerize by itself. Mutagenesis and protein-protein interaction studies demonstrate that some of the residues located at the HobA-DnaA I-II interface in the structure are necessary for complex formation. Introduction of selected mutations into H. pylori shows that the disruption of the interaction between HobA and DnaA is lethal for the bacteria. Remarkably, the DnaA binding site of HobA is conserved in DiaA from Escherichia coli, suggesting that the structure of the HobA/ DnaA complex represents a model for DnaA regulation in other Gram-negative bacteria. Our data, together with those from other studies, indicate that HobA could play a crucial scaffolding role during the initiation of replication in H. pylori by organizing the first step of DnaA oligomerization and attachment to oriC.DiaA ͉ isothermal titration calorimetry ͉ replication regulators ͉ X-ray crystallography

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Section: Discussionmentioning

confidence: 99%

“…In Bacillus subtilis, YabA is a negative regulator of initiation of chromosomal replication that downregulates initiation as part of a multimeric complex with DnaA and DnaN (19,20). B. subtilis DnaA activity is also controlled by the Soj (ParA) protein, which couples DNA replication with cell division (21,22).…”

mentioning

confidence: 99%

The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

Natrajan¹,

Noirot‐Gros

Zawilak-Pawlik

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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“…E. coli DnaA is converted from an active ATP bound form to an inactive ADP form after replication (14)(15)(16). A similar mechanism also operates in Bacillus subtilis (17). The initiators of iteron-carrying plasmids are inactivated by dimerization, the active form being monomers (18).…”

mentioning

confidence: 99%

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Venkova-Canova¹,

Srivastava²,

Chattoraj³

2006

Proc. Natl. Acad. Sci. U.S.A.

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The oriCII region that includes the initiator gene, rctB, can function as a plasmid in E. coli. Here we show that RctB suffices for the oriCII-based plasmid replication, and rctA in cis or trans reduces the plasmid copy number, thereby serving as a negative regulator. The inhibitory activity could be overcome by increasing the concentration of RctB, suggesting that rctA titrates the initiator. Purified RctB bound to a DNA fragment carrying rctA, confirming that the two can interact. Although rctA apparently works as a titrating site, it is nonetheless transcribed. We find that the transcription attenuates the inhibitory activity of the gene, presumably by interfering with RctB binding. RctB, in turn, repressed the rctA promoter and, thereby, could control its own titration by modulating the transcription of rctA. This control circuit appears to be a putative novel mechanism for homeostasis of initiator availability.O ur knowledge of DNA replication control in bacteria comes largely from studying the chromosome and plasmids of Escherichia coli (1, 2). In general, controlling proteins that initiate replication, initiator proteins, seems to be the central mechanism to regulate replication. A variety of mechanisms have been found to control the initiator.Transcriptional autoregulation of the initiator gene is common in plasmids with iterated initiator-binding sites (iterons), where the initiator serves as the repressor of its own synthesis (3). The E. coli initiator, DnaA, also is autoregulated (4). In addition, the dnaA promoter is inactivated by sequestration to the cell membrane for approximately one-third of the cell generation (5). More traditional modes of transcriptional control using repressors and activators also have been found in phage and plasmid RK2 (6, 7).Initiator translation is regulated by antisense RNAs in a variety of plasmids, the classic examples being plasmids R1 and pT181 (8, 9). The antisense RNA either blocks the ribosomebinding site of the initiator gene directly or of a regulatory peptide for the initiator gene (10).Most other mechanisms operate posttranslationally either by titrating or inactivating the initiator protein. In E. coli, there are Ϸ300 DnaA-binding sites scattered all over the chromosome (11,12). Titration of the initiator to these sites is believed to reduce availability of the protein after replication initiation, because new sites are created during replication elongation.Initiator inactivation is the most widely used mechanism to prevent premature replication reinitiation. The pT181 initiator is inactivated by covalent modification at the end of one round of replication (13). E. coli DnaA is converted from an active ATP bound form to an inactive ADP form after replication (14-16). A similar mechanism also operates in Bacillus subtilis (17). The initiators of iteron-carrying plasmids are inactivated by dimerization, the active form being monomers (18). Dimerization not only reduces monomer concentration, the dimers also participate in inactivating origins (19)(20)(21). Euk...

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“…Furthermore, two regulators of DnaA in E. coli, Hda described above and DiaA (40), are not present in S. aureus and B. subtilis. Instead, these Gram-positive bacteria contain YabA, which negatively regulates the initiation step of DNA replication by binding to DnaA and DnaN (41)(42)(43). Although DnaA is widely conserved in prokaryotes and is essential in both Gram-negative and Gram-positive bacteria, DnaA of B. subtilis and S. aureus is not interchangeable with E. coli DnaA (3,44).…”

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confidence: 99%

Rapid Exchange of Bound ADP on the Staphylococcus aureus Replication Initiation Protein DnaA

Kurokawa

Mizumura

Takaki

et al. 2009

Journal of Biological Chemistry

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In Escherichia coli, regulatory inactivation of the replication initiator DnaA occurs after initiation as a result of hydrolysis of bound ATP to ADP, but it has been unknown how DnaA is controlled to coordinate cell growth and chromosomal replication in Gram-positive bacteria such as Staphylococcus aureus. This study examined the roles of ATP binding and its hydrolysis in the regulation of the S. aureus DnaA activity. In vitro, S. aureus DnaA melted S. aureus oriC in the presence of ATP but not ADP by a mechanism independent of ATP hydrolysis. Unlike E. coli DnaA, binding of ADP to S. aureus DnaA was unstable. As a result, at physiological concentrations of ATP, ADP bound to S. aureus DnaA was rapidly exchanged for ATP, thereby regenerating the ability of DnaA to form the open complex in vitro. Therefore, we examined whether formation of ADP-DnaA participates in suppression of replication initiation in vivo. Induction of the R318H mutant of the AAA؉ sensor 2 protein, which has decreased intrinsic ATPase activity, caused over-initiation of chromosome replication in S. aureus, suggesting that formation of ADP-DnaA suppresses the initiation step in S. aureus. Together with the biochemical features of S. aureus DnaA, the weak ability to convert ATP-DnaA into ADP-DnaA and the instability of ADP-DnaA, these results suggest that there may be unidentified system(s) for reducing the cellular ratio of ATPDnaA to ADP-DnaA in S. aureus and thereby delaying the reinitiation of DNA replication.The initiation step of chromosome replication is an important set point for coordinating cell growth and chromosomal replication, and it is tightly regulated so that it takes place only once per cell cycle. DNA replication in bacteria starts when the cell mass reaches an appropriate size and at the appropriate time, which is controlled in part by the quantity and activity of initiator DnaA protein (1-3). In Escherichia coli, DnaA binds to 9-mer DnaA boxes in oriC, the origin of chromosome replication, and melts the duplex at AT-rich 13-mers adjacent to oriC. DnaA then directs loading of the DnaB replicative helicase, which depends on the DnaC helicase loader and priming by the DnaG primase. This is followed by the synthesis of DNA by the DNA polymerase III holoenzyme (4).DnaA is composed of four domains: N-terminal domains I and II are involved in DnaA oligomerization and DnaADnaB interaction, and they show a diversity of sequence among bacteria. Domain III contains AAAϩ motifs, and the C-terminal domain IV mediates DNA binding (3, 5). Biochemical and genetic studies in E. coli have revealed adenine nucleotide binding-mediated control of the DnaA activity (6 -11). Specifically, ATP-bound DnaA actively promotes replication initiation, whereas the ADP-bound form is inert (6, 7). ATP promotes the self-assembly of DnaA at replication origins via a conformational change to form a righthanded helical filament (8, 9). In E. coli, 70% to 80% of DnaA exists as the inactive ADP-bound form, and active ATPDnaA levels increase during the initiation ...

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Functional dissection of YabA, a negative regulator of DNA replication initiation in Bacillus subtilis

Cited by 77 publications

References 36 publications

The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria

Transcriptional inactivation of a regulatory site for replication of Vibrio cholerae chromosome II

Rapid Exchange of Bound ADP on the Staphylococcus aureus Replication Initiation Protein DnaA

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