The structural basis for dynamic DNA binding and bridging interactions which condense the bacterial centromere

Fisher, Gemma Lm; Pastrana, César L.; Higman, Victoria Ann; Koh, Alan; Taylor, James; Butterer, Annika; Craggs, Timothy D.; Sobott, Frank; Murray, Heath; Crump, Matthew P.; Moreno‐Herrero, Fernando; Dillingham, Mark S.

doi:10.7554/elife.28086

Cited by 68 publications

(116 citation statements)

References 74 publications

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“…The non-specific DNA-binding property of Bacillus ParB CTD was previously shown to condense DNA in vitro by magnetic tweezer assay (32,36,39) (Fig. 7A).…”

Section: Engineering a Lysine-rich Surface Into The Caulobacter Parb mentioning

confidence: 83%

“…Bacillus ParB condensed both non-parS DNA ( Fig. 7B) and parS DNA (32,36). On the contrary, Caulobacter ParB (WT) did not display any noticeable in vitro DNA condensation activity with either parS or non-parS DNA substrate at the tested concentration ( Fig.…”

Section: Engineering a Lysine-rich Surface Into The Caulobacter Parb mentioning

confidence: 93%

“…In addition to the NTD, the CTD of ParB from Bacillus subtilis also contributes to the formation of the ParB-DNA network (32,36,39). Bacillus ParB was reported to bind non-specific DNA to condense both parS and non-parS DNA in vitro; these activities were mediated by a positively charged lysinerich surface on the CTD (36,39). Whether the non-specific DNA-binding activity of the Bacillus ParB CTD is a shared feature among ParB orthologs is currently unknown, furthermore, the relationship between the in vitro DNA condensation and the in vivo spreading is not fully understood.…”

Section: Engineering a Lysine-rich Surface Into The Caulobacter Parb mentioning

confidence: 99%

“…7B). We performed experiments at 1 µM concentration of proteins and different forces using an identical setup and conditions described in experiments with Bacillus ParB (32,36). The extension of a tethered DNA was tracked, and any observation of a decrease in extension that was substantially larger than by the applied force alone is an indication of DNA condensation ( Fig.…”

Section: Engineering a Lysine-rich Surface Into The Caulobacter Parb mentioning

confidence: 99%

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A CTP-dependent gating mechanism enables ParB spreading on DNA

Jalal

Tran

Stevenson

et al. 2019

Preprint

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The tripartite ParA-ParB-parS complex ensures faithful chromosome segregation in the majority of bacterial species. ParB nucleates on a centromere-like parS site and spreads to neighboring DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the parS locus, hence the chromosome to each daughter cell. Here, we determine the cocrystal structure of a C-terminal domain truncated ParB-parS complex from Caulobacter crescentus, and show that its N-terminal domain adopts alternate conformations. The multiple conformations of the N-terminal domain might facilitate the spreading of ParB on the chromosome. Next, using ChIPseq we show that ParBs from different bacterial species exhibit variation in their intrinsic capability for spreading, and that the N-terminal domain is a determinant of this variability. Finally, we show that the C-terminal domain of Caulobacter ParB possesses no or weak non-specific DNA-binding activity. Engineered ParB variants with enhanced non-specific DNA-binding activity condense DNA in vitro but do not spread further than wild-type in vivo. Taken all together, our results emphasize the role of the N-terminal domain in ParB spreading and faithful chromosome segregation in Caulobacter crescentus. Proper chromosome segregation is essential in all domains of life. In two-thirds of known bacterial species, faithful chromosome segregation is mediated by the conserved ParA-ParB-parS system (1-10). This tripartite complex consists of a Walker-box ATPase ParA, a centromere-binding protein ParB, and a centromere-like DNA sequence parS. The parS site is the first DNA locus to be segregated after chromosome replication (2,5,11,12). ParB, a DNA-binding protein, nucleates on parS before binding to adjacent non-specific DNA to form a network of protein-DNA complexes. This nucleoprotein network interacts with ParA to partition the chromosome to each daughter cell. In Caulobacter crescentus, ParA forms a protein gradient emanating from the opposite pole of the cell to the ParB-parS complex (13-15). The DNA-bound ParB complexes stimulate the ATPase activity of ParA, causing the ParA gradient to retract, bringing the ParB-DNA complex to the opposite pole of the cell in a retreating gradient of ParA (15)(16)(17)(18)(19). In Caulobacter crescentus, ParA and ParB are essential for chromosome segregation and cell viability (3,13). In other bacterial species, engineered strains lacking ParB are still viable but have elevated numbers of anucleate cells due to defects in chromosome segregation (2,7,9,12,16,(20)(21)(22).The binding of multiple ParB molecules onto non-specific DNA after nucleation at parS (i.e. spreading) is a crucial event; bacterial cells harboring a nucleation-competent but spreadingdefective parB allele are impaired in plasmid/chromosome segregation (23-25). Spreading was first discovered for the F-plasmid-encoded SopB protein and the P1 plasmid-encoded ParB protein (26,27), and was subsequently found to be a general feature of many plasmid and chromo...

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“…The non-specific DNA-binding property of Bacillus ParB CTD was previously shown to condense DNA in vitro by magnetic tweezer assay (32,36,39) (Fig. 7A).…”

Section: Engineering a Lysine-rich Surface Into The Caulobacter Parb mentioning

confidence: 83%

Section: Engineering a Lysine-rich Surface Into The Caulobacter Parb mentioning

confidence: 93%

Section: Engineering a Lysine-rich Surface Into The Caulobacter Parb mentioning

confidence: 99%

Section: Engineering a Lysine-rich Surface Into The Caulobacter Parb mentioning

confidence: 99%

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A CTP-dependent gating mechanism enables ParB spreading on DNA

Jalal

Tran

Stevenson

et al. 2019

Preprint

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“…The formation of highly concentrated clusters of ParB relies on a strong ParB-parS interaction and two other interactions, ParB-ParB and ParB-nsDNA (Sanchez et al, 2015;Fisher et al, 2017). ParB mutants that do not propagate outside parS are impaired in partition activity and in cluster formation in vivo (Rodionov et al, 1999;Breier & Grossman, 2007).…”

Section: Discussionmentioning

confidence: 99%

A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids

Debaugny

Sanchez

Rech

et al. 2018

Molecular Systems Biology

113

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Chromosome and plasmid segregation in bacteria are mostly driven by ParABS systems. These DNA partitioning machineries rely on large nucleoprotein complexes assembled on centromere sites (parS). However, the mechanism of how a few parS‐bound ParB proteins nucleate the formation of highly concentrated ParB clusters remains unclear despite several proposed physico‐mathematical models. We discriminated between these different models by varying some key parameters in vivo using the F plasmid partition system. We found that “Nucleation & caging” is the only coherent model recapitulating in vivo data. We also showed that the stochastic self‐assembly of partition complexes (i) is a robust mechanism, (ii) does not directly involve ParA ATPase, (iii) results in a dynamic structure of discrete size independent of ParB concentration, and (iv) is not perturbed by active transcription but is by protein complexes. We refined the “Nucleation & caging” model and successfully applied it to the chromosomally encoded Par system of Vibrio cholerae, indicating that this stochastic self‐assembly mechanism is widely conserved from plasmids to chromosomes.

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DNA Segregation in Natural and Synthetic Minimal Systems

et al. 2019

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Faithful segregation of replicated genomes to dividing daughter cells is a major hallmark of cellular life and needs to be part of the future design of the robustly proliferating minimal cell. So far, the complexity of eukaryotic chromosome segregation machineries has limited their applicability to synthetic systems. Prokaryotic plasmid segregation machineries offer promising alternative tools for bottom‐up synthetic biology, with the first three‐component DNA segregation system being reconstituted a decade ago. In this review, the mechanisms underlying DNA segregation in prokaryotes, with a particular focus on segregation of plasmids and chromosomal replication origins are reviewed, along with a brief discussion of archaeal and eukaryotic systems. In addition, this review shows how in vitro reconstitution has allowed deeper insights into these processes and discusses possible applications of these machineries for a minimal synthetic segrosome as well as the challenge of its coupling to a minimal replisome.

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The structural basis for dynamic DNA binding and bridging interactions which condense the bacterial centromere

Cited by 68 publications

References 74 publications

A CTP-dependent gating mechanism enables ParB spreading on DNA

A CTP-dependent gating mechanism enables ParB spreading on DNA

A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids

DNA Segregation in Natural and Synthetic Minimal Systems

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