Beyond DnaA: The Role of DNA Topology and DNA Methylation in Bacterial Replication Initiation

Donczew, Rafał; Zakrzewska‐Czerwińska, Jolanta; Zawilak-Pawlik, Anna

doi:10.1016/j.jmb.2014.04.009

Cited by 19 publications

(15 citation statements)

References 163 publications

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“…Replication initiation is regulated by a complex combination of factors, but it is ultimately dependent on two parameters, chromosome structure at the origin and binding of activated (ATP-bound) DnaA protein [reviewed in 1, 2, 17]. To test whether tethering blocked initiation by effects on DnaA, we tethered cells carrying a deletion in one of three DnaA negative regulators, SeqA, an protein that binds oriC and inhibits DnaA binding [5, 11, 12], Hda, a replisome-associated protein that stimulates DnaA-ATP hydrolysis [18, 19], or datA , a cluster of DnaA binding sites that also stimulates DnaA-ATP hydrolysis [20].…”

Section: Resultsmentioning

confidence: 99%

DNA Replication Initiation Is Blocked by a Distant Chromosome–Membrane Attachment

et al. 2015

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Summary Although it has been recognized for several decades that chromosome structure regulates the capacity of replication origins to initiate, very little is known about how or if cells actively regulate structure to direct initiation [1–3]. We report that a localized inducible protein tether between the chromosome and cell membrane in E. coli cells imparts a rapid and complete block to replication initiation. Tethers, composed of a trans-membrane and transcription repressor fusion protein bound to an array of operator sequences, can be placed up to one megabase from the origin with no loss of penetrance. Tether-induced initiation blocking has no effect on elongation at pre-existing replication forks and does not cause cell or DNA damage. Whole-genome and site-specific fluorescent DNA labeling in tethered cells indicates that global nucleoid structure and chromosome organization are disrupted. Gene expression patterns, assayed by RNA sequencing shows that tethering induces global supercoiling changes, which are likely incompatible with replication initiation. Parallels between tether-induced initiation blocking and rifampicin treatment, and the role of programmed changes in chromosome structure in replication control are discussed.

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Section: Resultsmentioning

confidence: 99%

DNA Replication Initiation Is Blocked by a Distant Chromosome–Membrane Attachment

et al. 2015

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“…However, in the initiation process not only the sequence specificity of the dnaA boxes and binding of DnaA but also the topology of the DNA has been shown to be important (57–63). For example, it is known that negative supercoiling of the DNA in oriC promotes DNA unwinding (64).…”

Section: Discussionmentioning

confidence: 99%

“…Also, loop formation within an unstable region in oriC has been suggested to initiate DNA unwinding and enable interaction between distantly separated oligomers of DnaA (53,60,62,65). Nucleoid-associated proteins such as Fis, IHF and HU have been proposed to modulate oriC topology for regulation of replication initiation (in addition to DnaA) (57,62,63,66–68), but in this context the role of SeqA remains to be explored. A positive effect of SeqA on initiation has been found at low DnaA concentrations in vitro (69).…”

Section: Discussionmentioning

confidence: 99%

Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome

Helgesen

Fossum-Raunehaug

Sætre

et al. 2015

Nucleic Acids Research

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The Escherichia coli SeqA protein binds to newly replicated, hemimethylated DNA behind replication forks and forms structures consisting of several hundred SeqA molecules bound to about 100 kb of DNA. It has been suggested that SeqA structures either direct the new sister DNA molecules away from each other or constitute a spacer that keeps the sisters together. We have developed an image analysis script that automatically measures the distance between neighboring foci in cells. Using this tool as well as direct stochastic optical reconstruction microscopy (dSTORM) we find that in cells with fluorescently tagged SeqA and replisome the sister SeqA structures were situated close together (less than about 30 nm apart) and relatively far from the replisome (on average 200–300 nm). The results support the idea that newly replicated sister molecules are kept together behind the fork and suggest the existence of a stretch of DNA between the replisome and SeqA which enjoys added stabilization. This could be important in facilitating DNA transactions such as recombination, mismatch repair and topoisomerase activity. In slowly growing cells without ongoing replication forks the SeqA protein was found to reside at the fully methylated origins prior to initiation of replication.

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“…In E. coli , where initiation of bacterial chromosomal replication has been extensively studied, binding of multiple DnaA initiator proteins (∼53 kD) to sites within the 245 bp oriC DNA sequence leads to assembly of a large multi-protein DNA complex (1,9,10). The DnaA–OriC complex mediates initial melting of origin DNA within an A-T rich segment of the origin (1,11,12); the resulting single-stranded DNA is bound and stabilized by an oligomeric form of DnaA (4,13). Melted DNA at the origin serves as the entry point for the replicative helicase, and additional events lead to establishment of the replisome (14).…”

Section: Introductionmentioning

confidence: 99%

The replication initiator of the cholera pathogen's second chromosome shows structural similarity to plasmid initiators

et al. 2016

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The conserved DnaA-oriC system is used to initiate replication of primary chromosomes throughout the bacterial kingdom; however, bacteria with multipartite genomes evolved distinct systems to initiate replication of secondary chromosomes. In the cholera pathogen, Vibrio cholerae, and in related species, secondary chromosome replication requires the RctB initiator protein. Here, we show that RctB consists of four domains. The structure of its central two domains resembles that of several plasmid replication initiators. RctB contains at least three DNA binding winged-helix-turn-helix motifs, and mutations within any of these severely compromise biological activity. In the structure, RctB adopts a head-to-head dimeric configuration that likely reflects the arrangement in solution. Therefore, major structural reorganization likely accompanies complex formation on the head-to-tail array of binding sites in oriCII. Our findings support the hypothesis that the second Vibrionaceae chromosome arose from an ancestral plasmid, and that RctB may have evolved additional regulatory features.

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Beyond DnaA: The Role of DNA Topology and DNA Methylation in Bacterial Replication Initiation

Cited by 19 publications

References 163 publications

DNA Replication Initiation Is Blocked by a Distant Chromosome–Membrane Attachment

DNA Replication Initiation Is Blocked by a Distant Chromosome–Membrane Attachment

Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome

The replication initiator of the cholera pathogen's second chromosome shows structural similarity to plasmid initiators

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