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1.
Proc Natl Acad Sci U S A ; 119(41): e2204042119, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36206370

RESUMO

SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.


Assuntos
Bacillus subtilis , Cromossomos Bacterianos , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos
2.
Cell Rep ; 40(9): 111273, 2022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-36044845

RESUMO

Chromosomes readily unlink and segregate to daughter cells during cell division, highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes promote DNA organization by loop extrusion. In most bacteria, chromosome folding initiates at dedicated start sites marked by the ParB/parS partition complexes. Whether SMC complexes recognize a specific DNA structure in the partition complex or a protein component is unclear. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate foreign chromosomes. Using chimeric proteins and chemical cross-linking, we find that ParB directly binds the Smc subunit. We map an interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex, presumably to initiate DNA loop extrusion.


Assuntos
Proteínas de Bactérias , Proteínas de Ciclo Celular , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/metabolismo , DNA/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo
3.
Sci Adv ; 7(41): eabj2854, 2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34613769

RESUMO

Three-component ParABS systems are widely distributed factors for plasmid partitioning and chromosome segregation in bacteria. ParB acts as adaptor protein between the 16­base pair centromeric parS DNA sequences and the DNA segregation proteins ParA and Smc (structural maintenance of chromosomes). Upon cytidine triphosphate (CTP) and parS DNA binding, ParB dimers form DNA clamps that spread onto parS-flanking DNA by sliding, thus assembling the so-called partition complex. We show here that CTP hydrolysis is essential for efficient chromosome segregation by ParABS but largely dispensable for Smc recruitment. Our results suggest that CTP hydrolysis contributes to partition complex assembly via two mechanisms. It promotes ParB unloading from DNA to limit the extent of ParB spreading, and it recycles off-target ParB clamps to allow for parS retargeting, together superconcentrating ParB near parS. We also propose a model for clamp closure involving a steric clash when binding ParB protomers to opposing parS half sites.

4.
Elife ; 102021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34346312

RESUMO

SMC complexes are widely conserved ATP-powered DNA-loop-extrusion motors indispensable for organizing and faithfully segregating chromosomes. How SMC complexes translocate along DNA for loop extrusion and what happens when two complexes meet on the same DNA molecule is largely unknown. Revealing the origins and the consequences of SMC encounters is crucial for understanding the folding process not only of bacterial, but also of eukaryotic chromosomes. Here, we uncover several factors that influence bacterial chromosome organization by modulating the probability of such clashes. These factors include the number, the strength, and the distribution of Smc loading sites, the residency time on the chromosome, the translocation rate, and the cellular abundance of Smc complexes. By studying various mutants, we show that these parameters are fine-tuned to reduce the frequency of encounters between Smc complexes, presumably as a risk mitigation strategy. Mild perturbations hamper chromosome organization by causing Smc collisions, implying that the cellular capacity to resolve them is limited. Altogether, we identify mechanisms that help to avoid Smc collisions and their resolution by Smc traversal or other potentially risky molecular transactions.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos , Cromossomos Bacterianos , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo
5.
Cell Rep ; 14(8): 2003-16, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26904953

RESUMO

Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome-presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc.


Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromossomos Bacterianos/química , DNA Primase/metabolismo , DNA Bacteriano/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Divisão Celular , Segregação de Cromossomos , Cromossomos Bacterianos/ultraestrutura , DNA Primase/genética , DNA Bacteriano/genética , Hidrólise , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Transporte Proteico , Alinhamento de Sequência
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