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

Debaugny, Roxanne E; Sanchez, Aurore; Rech, Jérôme; Labourdette, Delphine; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Boudsocq, François; Leberre, Véronique Anton; Walter, Jean‐Charles; Bouet, Jean‐Yves

doi:10.15252/msb.20188516

Cited by 66 publications

(127 citation statements)

References 61 publications

(129 reference statements)

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“…Our novel approach allowed us to visualize the dynamic nature of the ParB binding with evidence for fast and continuous exchange of proteins with the surrounding media. This dynamic behavior is consistent with results described not only for BsParB (Graham et al, 2014) (Taylor et al, 2015) (Song et al, 2017) (Fisher et al, 2017), but also for E. coli ParB (Sanchez et al, 2015) (Le Gall et al, 2016), plasmid ParB (Debaugny et al, 2018), and for other proteins, at the single molecule level (Gibb et al, 2014). Implementation of a multilaminar flow exchange system to the combined MT-TIRF setup provided a measurement of binding and unbinding reactions in the presence of the free CTD leading to the conclusion that it only affects binding and unbinding of ParB in a buffer supplemented with EDTA.…”

Section: Discussionsupporting

confidence: 91%

ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

Madariaga-Marcos

Pastrana

Fisher

et al. 2018

Preprint

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Bacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. In our previous work (Fisher et al., 2017), we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks. However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualize ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds nonspecifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerization with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks. Significance StatementUsing combined Magnetic Tweezers and TIRF microscopy we show that the CTD of ParB blocks ParB network formation by heterodimerization with the full-length protein, which remains bound to the DNA.

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Section: Discussionsupporting

confidence: 91%

ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

Madariaga-Marcos

Pastrana

Fisher

et al. 2018

Preprint

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“…Analysis of ParB complexes using 2D PALM reveals ParB-dense regions within clusters that correlate to the number of ParB enrichment zones along adjacent parS sites. In line with a current study on ParB cluster-assembly in V. cholerae 61 , we suggest that these subclusters derive from independent nucleation and caging events, which merge into one ParB-macrocomplex per oriC in C. glutamicum . Presence of a single parS site leads to formation of almost globular ParB densities.…”

Section: Discussionsupporting

confidence: 90%

Chromosome organization by a conserved condensin-ParB system in the actinobacteriumCorynebacterium glutamicum

Boehm

Giacomelli

Schmidt

et al. 2019

Preprint

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20Higher-order chromosome folding and segregation is tightly regulated in all domains 21 of life. In bacteria, details on nucleoid organization regulatory mechanisms and function 22 remains poorly characterized, especially in non-model species. Here, we investigate 23 the role of DNA partitioning protein ParB and condensin complexes, two key players 24 in bacterial chromosome structuring, in the actinobacterium Corynebacterium 25 glutamicum. Chromosome conformation capture reveals SMC-mediated long-range 26 interactions around ten centromere-like parS sites clustered at the replication origin 27 (oriC). At least one oriC-proximal parS site is necessary for a reliable chromosome 28 segregation. Using a combination of chromatin immunoprecipitation and 29 photoactivated single molecule localization microscopy evidences the formation of 30 distinct ParB-nucleoprotein subclusters in dependence of parS numbers. We further 31 identified and functionally characterized two condensin paralogs. Whereas 32 SMC/ScpAB complexes are loaded via ParB at parS sites mediating chromosomal 33 inter-arm contacts like in Bacillus subtilis, the MukBEF-like SMC complex MksBEFG 34 does not contribute to chromosomal DNA-folding. Rather, the MksBEFG complex is 35 involved in plasmid maintenance and interacts with the polar oriC-tethering factor 36 DivIVA. These data complement current models of ParB-SMC/ScpAB crosstalk, while 37 showing that some condensin complexes evolved functions uncoupled from 38 chromosome folding. 39 Keywords 40 ParB, parS, SMC, MksB, condensin, actinobacteria, chromosome conformation 41 capture, chromatin immunoprecipitation, super-resolution microscopy, DNA 42 segregation 43 44 can cause severe DNA segregation phenotypes 18,19 . To date, only few studies 71 investigated the impact of chromosomal parS localization on DNA-segregation and 72 folding 18-22 . 73 In addition to ParABS systems, most bacteria harbor condensin complexes, members 74 of the structural maintenance of chromosomes (SMC) family of proteins found in all 75 kingdoms of life 23 . In standard model organisms, condensins are equally essential for 76 faithful chromosome segregation by compacting DNA into separate nucleoids 24-26 . The 77 SMC/ScpAB (structural maintenance of chromosomes) complex is well-studied in B. 78 subtilis, where it consists of two large SMC subunits and the kleisin ScpA associated 79 with dimeric accessory protein ScpB that assemble into a ring-like structure 27 . A recent 80 study suggests progressive extrusion of condensin-encircled DNA loops upon 81 conformational changes in the SMC subunit, which leads to a gradual size increase of 82 trapped DNA molecules 28 . The active process(es) driving DNA extrusion 29,30 allow(s) 83 for translocation along the chromosome with velocities of around 50 Kb/ min 31 , and 84 depend(s) on the ATPase activity of SMC 32 . To be loaded on parS sites, SMC/ScpAB 85 complexes necessitate ParB 20,22,33,34 . They redistribute to distant chromosomal 86 regions, promoting topological changes, and notab...

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“…The centromere parS is the first DNA locus to be segregated following chromosome replication 8,9,14,15 . ParB specifically nucleates on parS before spreading outwards to the flanking DNA and bridges/cages DNA together to form a nucleoprotein network in vivo [16][17][18][19][20][21][22][23] . This nucleoprotein complex recruits SMC to disentangle and organize replicated DNA 3,11,13,24,25 .…”

mentioning

confidence: 99%

ParB spreading on DNA requires cytidine triphosphatein vitro

Jalal

Tran

2019

Preprint

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In all living organisms, it is essential to transmit genetic information faithfully to the next generation. The SMC-ParAB-parS system is widely employed for chromosome segregation in bacteria. A DNA-binding protein ParB nucleates on parS sites and must associate with neighboring DNA, a process known as spreading, to enable efficient chromosome segregation. Despite its importance, how the initial few ParB molecules nucleating at parS sites recruit hundreds of further ParB to spread is not fully understood. Here, we reconstitute a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and show that CTP is required for spreading. We further show that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC.Faithful chromosome segregation is essential in all domains of life if daughter cells are each to inherit the full set of genetic information. The SMC-ParAB-parS complex is widely employed for chromosome segregation in bacteria 1-13 . The centromere parS is the first DNA locus to be segregated following chromosome replication 8,9,14,15 . ParB specifically nucleates on parS before spreading outwards to the flanking DNA and bridges/cages DNA together to form a nucleoprotein network in vivo [16][17][18][19][20][21][22][23] . This nucleoprotein complex recruits SMC to disentangle and organize replicated DNA 3,11,13,24,25 . ParB-parS also interacts with an ATPase ParA to power the segregation of replicated chromosomes [26][27][28][29][30] . Engineered strains harboring a nucleation-competent but spreading-defective mutant of parB are either unviable 10 or have elevated number of anucleate cells 4,7,8,15,[31][32][33][34] . Despite the importance of spreading for proper chromosome segregation, the mechanism by which a few parS-bound ParB can recruit hundreds more ParB molecules to the vicinity of parS to assemble a high molecular-weight nucleoprotein complex is not fully understood.Since the first report in 1995 35 , ParB spreading has been observed in vivo by chromatin immunoprecipitation in multiple bacterial species 12,[15][16][17]19,36 . The nucleation of ParB on parS has also been demonstrated in vitro 4,10,16,17,20,[37][38][39] , however parS-dependent ParB spreading has resisted biochemical reconstitution [17][18][19]40,41 . Unsuccessful attempts to reconstitute spreading in vitro suggests that additional factors might be missing. Recently, works by Soh et al (2019) and Osorio-Valeriano et al (2019) on Bacillus subtilis and Myxococcus xanthus ParB, respectively, showed that ParB binds and hydrolyzes cytidine triphosphate (CTP) to cytidine diphosphate (CDP), and that CTP modulates the binding affinity of ParB to parS 42,43 . A co-crystal structure of Bacillus ParB with CDP and that of a Myxococcus ParB-li...

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

Cited by 66 publications

References 61 publications

ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

ParB dynamics and the critical role of the CTD in DNA condensation unveiled by combined force-fluorescence measurements

Chromosome organization by a conserved condensin-ParB system in the actinobacteriumCorynebacterium glutamicum

ParB spreading on DNA requires cytidine triphosphatein vitro

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