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A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

ABSTRACT: Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP-PNP-DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP-PNP-DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.

SUBMITTER: Saxena R

PROVIDER: S-EPMC7145717 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

Saxena Rahul R Stanley Christopher B CB Kumar Pankaj P Kumar Pankaj P Cuneo Matthew J MJ Patil Digvijay D Jha Jyoti J Weiss Kevin L KL Chattoraj Dhruba K DK Crooke Elliott E

Nucleic acids research 20200101 1

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of Dna ...[more]

PMID: 31665475

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Project description:UnlabelledIt is not known how diverse bacteria regulate chromosome replication. Based on Escherichia coli studies, DnaA initiates replication and the homolog of DnaA (Hda) inactivates DnaA using the RIDA (regulatory inactivation of DnaA) mechanism that thereby prevents extra chromosome replication cycles. RIDA may be widespread, because the distantly related Caulobacter crescentus homolog HdaA also prevents extra chromosome replication (J. Collier and L. Shapiro, J Bacteriol 191:5706-5715, 2009, http://dx.doi.org/10.1128/JB.00525-09). To further study the HdaA/RIDA mechanism, we created a C. crescentus strain that shuts off hdaA transcription and rapidly clears HdaA protein. We confirm that HdaA prevents extra replication, since cells lacking HdaA accumulate extra chromosome DNA. DnaA binds nucleotides ATP and ADP, and our results are consistent with the established E. coli mechanism whereby Hda converts active DnaA-ATP to inactive DnaA-ADP. However, unlike E. coli DnaA, C. crescentus DnaA is also regulated by selective proteolysis. C. crescentus cells lacking HdaA reduce DnaA proteolysis in logarithmically growing cells, thereby implicating HdaA in this selective DnaA turnover mechanism. Also, wild-type C. crescentus cells remove all DnaA protein when they enter stationary phase. However, cells lacking HdaA retain stable DnaA protein even when they stop growing in nutrient-depleted medium that induces complete DnaA proteolysis in wild-type cells. Additional experiments argue for a distinct HdaA-dependent mechanism that selectively removes DnaA prior to stationary phase. Related freshwater Caulobacter species also remove DnaA during entry to stationary phase, implying a wider role for HdaA as a novel component of programed proteolysis.ImportanceBacteria must regulate chromosome replication, and yet the mechanisms are not completely understood and not fully exploited for antibiotic development. Based on Escherichia coli studies, DnaA initiates replication, and the homolog of DnaA (Hda) inactivates DnaA to prevent extra replication. The distantly related Caulobacter crescentus homolog HdaA also regulates chromosome replication. Here we unexpectedly discovered that unlike the E. coli Hda, the C. crescentus HdaA also regulates DnaA proteolysis. Furthermore, this HdaA proteolysis acts in logarithmically growing and in stationary-phase cells and therefore in two very different physiological states. We argue that HdaA acts to help time chromosome replications in logarithmically growing cells and that it is an unexpected component of the programed entry into stationary phase.

Dataset Information

A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

Publications

A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

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