Multiple DNA Binding Proteins Contribute to Timing of Chromosome Replication in E. coli

Riber, Leise; Frimodt-Møller, Jakob; Charbon, Godefroid; Løbner-Olesen, Anders

doi:10.3389/fmolb.2016.00029

Cited by 41 publications

(85 citation statements)

References 91 publications

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“…Even when cells grow rapidly and the copy number of oriC increases to more than two per cell, initiation occurs at sister oriC regions simultaneously only once at a specific time during the cell cycle. As such, the time of initiation at oriC is regulated and re-initiation during the same cell cycle is strictly repressed ( Skarstad and Katayama, 2013 ; Wolański et al, 2015 ; Riber et al, 2016 ).…”

Section: Introductionmentioning

confidence: 99%

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The DnaA Cycle in Escherichia coli: Activation, Function and Inactivation of the Initiator Protein

2017

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This review summarizes the mechanisms of the initiator protein DnaA in replication initiation and its regulation in Escherichia coli. The chromosomal origin (oriC) DNA is unwound by the replication initiation complex to allow loading of DnaB helicases and replisome formation. The initiation complex consists of the DnaA protein, DnaA-initiator-associating protein DiaA, integration host factor (IHF), and oriC, which contains a duplex-unwinding element (DUE) and a DnaA-oligomerization region (DOR) containing DnaA-binding sites (DnaA boxes) and a single IHF-binding site that induces sharp DNA bending. DiaA binds to DnaA and stimulates DnaA assembly at the DOR. DnaA binds tightly to ATP and ADP. ATP-DnaA constructs functionally different sub-complexes at DOR, and the DUE-proximal DnaA sub-complex contains IHF and promotes DUE unwinding. The first part of this review presents the structures and mechanisms of oriC-DnaA complexes involved in the regulation of replication initiation. During the cell cycle, the level of ATP-DnaA level, the active form for initiation, is strictly regulated by multiple systems, resulting in timely replication initiation. After initiation, regulatory inactivation of DnaA (RIDA) intervenes to reduce ATP-DnaA level by hydrolyzing the DnaA-bound ATP to ADP to yield ADP-DnaA, the inactive form. RIDA involves the binding of the DNA polymerase clamp on newly synthesized DNA to the DnaA-inactivator Hda protein. In datA-dependent DnaA-ATP hydrolysis (DDAH), binding of IHF at the chromosomal locus datA, which contains a cluster of DnaA boxes, results in further hydrolysis of DnaA-bound ATP. SeqA protein inhibits untimely initiation at oriC by binding to newly synthesized oriC DNA and represses dnaA transcription in a cell cycle dependent manner. To reinitiate DNA replication, ADP-DnaA forms oligomers at DnaA-reactivating sequences (DARS1 and DARS2), resulting in the dissociation of ADP and the release of nucleotide-free apo-DnaA, which then binds ATP to regenerate ATP-DnaA. In vivo, DARS2 plays an important role in this process and its activation is regulated by timely binding of IHF to DARS2 in the cell cycle. Chromosomal locations of DARS sites are optimized for the strict regulation for timely replication initiation. The last part of this review describes how DDAH and DARS regulate DnaA activity.

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Section: Introductionmentioning

confidence: 99%

“…Escherichia coli cells have at least three regulatory systems for DnaA activity ( Skarstad and Katayama, 2013 ; Riber et al, 2016 ). In regulatory inactivation of DnaA (RIDA), ATP-DnaA is inactivated in a negative-feedback manner coupled to DNA replication ( Katayama et al, 2010 ).…”

Section: Introductionmentioning

confidence: 99%

The DnaA Cycle in Escherichia coli: Activation, Function and Inactivation of the Initiator Protein

2017

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“…The cell cycle-coordinated initiation of chromosomal replication is sustained by multiple regulatory systems that affect cellular DnaA activity (1,4,28). After ATP-DnaA induces replication initiation, DnaA-ATP is hydrolyzed by the complex of the AAAϩ domain-containing Hda protein and the DNAloaded ␤-clamp subunit of the DNA polymerase III holoenzyme, yielding initiation-inactive ADP-DnaA (1, 29 -31).…”

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confidence: 99%

“…The datA locus (991 bp) includes a 262-bp region containing two known DnaA-box sequences (DnaA boxes 2 and 3) and an IBS, which is essential for repression of untimely initiation ( Fig. 1A) (25)(26)(27)(28). DNA-footprinting experiments suggest the presence of two further sites within the 262-bp datA region that bind DnaA (herein described as DnaA boxes 6 and 7) ( Fig.…”

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confidence: 99%

Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis

Kasho

Tanaka²,

Sakai

et al. 2017

Journal of Biological Chemistry

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Edited by Xiao-Fan WangTimely initiation of replication in Escherichia coli requires functional regulation of the replication initiator, ATP-DnaA. The cellular level of ATP-DnaA increases just before initiation, after which its level decreases through hydrolysis of DnaAbound ATP, yielding initiation-inactive ADP-DnaA. Previously, we reported a novel DnaA-ATP hydrolysis system involving the chromosomal locus datA and named it datA-dependent DnaA-ATP hydrolysis (DDAH). The datA locus contains a binding site for a nucleoid-associating factor integration host factor (IHF) and a cluster of three known DnaA-binding sites, which are important for DDAH. However, the mechanisms underlying the formation and regulation of the datA-IHF⅐DnaA complex remain unclear. We now demonstrate that a novel DnaA box within datA is essential for ATP-DnaA complex formation and DnaA-ATP hydrolysis. Specific DnaA residues, which are important for interaction with bound ATP and for head-to-tail inter-DnaA interaction, were also required for ATP-DnaA-specific oligomer formation on datA. Furthermore, we show that negative DNA supercoiling of datA stabilizes ATP-DnaA oligomers, and stimulates datA-IHF interaction and DnaA-ATP hydrolysis. Relaxation of DNA supercoiling by the addition of novobiocin, a DNA gyrase inhibitor, inhibits datA function in cells. On the basis of these results, we propose a mechanistic model of datA-IHF⅐DnaA complex formation and DNA supercoiling-dependent regulation for DDAH.In Escherichia coli, the ATP-bound DnaA protein (ATPDnaA) 3 has an essential role in assembly of initiation complexes on the chromosomal replication origin oriC (1-5). oriC consists of an AT-rich duplex-unwinding element (DUE) and a DnaA oligomerization region that contains two subregions with oppositely oriented clusters of 9-bp DnaA-binding sites (DnaA boxes) (see Fig. 1A). DnaA boxes R1 and R4 at the outer edges of the DnaA oligomerization region are oriented oppositely and have the highest affinities for DnaA of the sequences in this region. Cooperative binding of ATP-DnaA molecules to lower affinity sites in each subregion of oriC results in formation of left-half and right-half DnaA subcomplexes (6 -8). The lefthalf subcomplex binds to the region of oriC that contains the DUE and executes unwinding of DNA within the element (6, 9). IHF, a nucleoid-associated protein (NAP), binds to a specific IHF-binding site (IBS; see Fig. 1A) within the left-half subregion of oriC and bends DNA sharply (ϳ180°), enhancing ATP-DnaA binding and duplex unwinding (9, 10). The DnaA subcomplexes also interact with a pair of DnaB helicases for loading of those to the single-stranded DNA (9,11,12).DnaA consists of four domains (3). The N-terminal domain I has a DnaB-binding site (12). Domain II is a flexible linker (12). Domain III is the AAAϩ domain that has motifs for binding and hydrolysis of ATP (Walker A and B and Sensor I and II) and for interaction between DnaA protomers (arginine-finger, Box VII, AID-1, and AID-2) (see Fig. 1B) (6, 13-16). DnaA argininefin...

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“…Most in vivo replication systems rely on DNA binding proteins for correct function, whether by recruiting other proteins involved in replication, or simply inhibiting double strand formation [36,37] . In some systems, it can even be used to prime the replication itself [38] .…”

Section: Cell Display For the Engineering Of Dna/xna Binding Proteinsmentioning

confidence: 99%

Towards XNA molecular biology: Bacterial cell display as a robust and versatile platform for the engineering of low affinity ligands and enzymes

Csibra

Renders

Pinheiro

2020

Preprint

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Although directed evolution has been remarkably successful at expanding the chemical and functional boundaries of biology, it is limited by the robustness and flexibility of available selection platforms -traditionally designed around a single desired function with limited scope for alternative applications. We report SNAP as a quantitative reporter for bacterial cell display, which enabled fast troubleshooting and systematic development of the selection platform. In addition, we demonstrate that even weak interactions between displayed proteins and nucleic acids can be harnessed towards specific labelling of bacterial cells, allowing functional characterisation of DNA binding proteins and enzymes. Together, this establishes bacterial display as a viable route towards the systematic engineering of all ligands and enzymes required for the development of XNA molecular biology.

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Multiple DNA Binding Proteins Contribute to Timing of Chromosome Replication in E. coli

Cited by 41 publications

References 91 publications

The DnaA Cycle in Escherichia coli: Activation, Function and Inactivation of the Initiator Protein

The DnaA Cycle in Escherichia coli: Activation, Function and Inactivation of the Initiator Protein

Cooperative DnaA Binding to the Negatively Supercoiled datA Locus Stimulates DnaA-ATP Hydrolysis

Towards XNA molecular biology: Bacterial cell display as a robust and versatile platform for the engineering of low affinity ligands and enzymes

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