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SMC condensin entraps chromosomal DNA by an ATP hydrolysis dependent loading mechanism in Bacillus subtilis.

Wilhelm L, Bürmann F, Minnen A, Shin HC, Toseland CP, Oh BH, Gruber S - Elife (2015)

Bottom Line: Exclusively species of Smc-ScpA, which were previously cross-linked into covalent rings, remained associated with chromosomal DNA.DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted, implying that most Smc-ScpAB is loaded onto the chromosome at parS sites near the replication origin.We furthermore report a physical interaction between native Smc-ScpAB and chromosomal DNA fragments.

View Article: PubMed Central - PubMed

Affiliation: Chromosome Organization and Dynamics, Max Planck Institute of Biochemistry, Martinsried, Germany.

ABSTRACT
Smc-ScpAB forms elongated, annular structures that promote chromosome segregation, presumably by compacting and resolving sister DNA molecules. The mechanistic basis for its action, however, is only poorly understood. Here, we have established a physical assay to determine whether the binding of condensin to native chromosomes in Bacillus subtilis involves entrapment of DNA by the Smc-ScpAB ring. To do so, we have chemically cross-linked the three ring interfaces in Smc-ScpAB and thereafter isolated intact chromosomes under protein denaturing conditions. Exclusively species of Smc-ScpA, which were previously cross-linked into covalent rings, remained associated with chromosomal DNA. DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted, implying that most Smc-ScpAB is loaded onto the chromosome at parS sites near the replication origin. We furthermore report a physical interaction between native Smc-ScpAB and chromosomal DNA fragments.

No MeSH data available.


Growth of smc(Cys) mutants.Overnight cultures of strains (BSG1002, BSG1007, BSG1782, BSG1850 and BSG1783) were serially diluted and spotted as described before (Figure 2—figure supplement 1). Strains were wild-type (‘WT’), harboured the smc deletion (‘Δ’) or cysteine mutations for cross-linking the Smc-ScpA ring (‘Cys’). In addition, scpB (‘ΔscpB’) or parB (‘ΔparB’) were deleted where indicated.DOI:http://dx.doi.org/10.7554/eLife.06659.011
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fig4s1: Growth of smc(Cys) mutants.Overnight cultures of strains (BSG1002, BSG1007, BSG1782, BSG1850 and BSG1783) were serially diluted and spotted as described before (Figure 2—figure supplement 1). Strains were wild-type (‘WT’), harboured the smc deletion (‘Δ’) or cysteine mutations for cross-linking the Smc-ScpA ring (‘Cys’). In addition, scpB (‘ΔscpB’) or parB (‘ΔparB’) were deleted where indicated.DOI:http://dx.doi.org/10.7554/eLife.06659.011

Mentions: What other factors might be required for the loading of condensin onto DNA? The ScpB subunit forms homodimers that bind in an asymmetric manner to the central region of a single ScpA monomer. It thus is in close proximity of the Smc ATPase domains. Together with ScpA it putatively plays a role in the regulation of the Smc ATPase activity (Kamada et al., 2013). Its precise molecular function, however, is not clear yet. To test whether ScpB is involved in the association of Smc–ScpA rings with chromosomes we combined the cysteine mutations in Smc and ScpA with an scpB in-frame deletion (Figure 4—figure supplement 1). Ring formation was only mildly affected by the absence of ScpB as judged by BMOE cross-linking and in-gel fluorescence detection (Figure 4A) (Bürmann et al., 2013). However, Smc complexes lacking ScpB subunits failed to entrap chromosomes altogether demonstrating that ScpB is absolutely required for loading of prokaryotic condensin onto chromosomal DNA.10.7554/eLife.06659.010Figure 4.ScpB and ParB are essential for efficient DNA entrapment by Smc complexes.


SMC condensin entraps chromosomal DNA by an ATP hydrolysis dependent loading mechanism in Bacillus subtilis.

Wilhelm L, Bürmann F, Minnen A, Shin HC, Toseland CP, Oh BH, Gruber S - Elife (2015)

Growth of smc(Cys) mutants.Overnight cultures of strains (BSG1002, BSG1007, BSG1782, BSG1850 and BSG1783) were serially diluted and spotted as described before (Figure 2—figure supplement 1). Strains were wild-type (‘WT’), harboured the smc deletion (‘Δ’) or cysteine mutations for cross-linking the Smc-ScpA ring (‘Cys’). In addition, scpB (‘ΔscpB’) or parB (‘ΔparB’) were deleted where indicated.DOI:http://dx.doi.org/10.7554/eLife.06659.011
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4442127&req=5

fig4s1: Growth of smc(Cys) mutants.Overnight cultures of strains (BSG1002, BSG1007, BSG1782, BSG1850 and BSG1783) were serially diluted and spotted as described before (Figure 2—figure supplement 1). Strains were wild-type (‘WT’), harboured the smc deletion (‘Δ’) or cysteine mutations for cross-linking the Smc-ScpA ring (‘Cys’). In addition, scpB (‘ΔscpB’) or parB (‘ΔparB’) were deleted where indicated.DOI:http://dx.doi.org/10.7554/eLife.06659.011
Mentions: What other factors might be required for the loading of condensin onto DNA? The ScpB subunit forms homodimers that bind in an asymmetric manner to the central region of a single ScpA monomer. It thus is in close proximity of the Smc ATPase domains. Together with ScpA it putatively plays a role in the regulation of the Smc ATPase activity (Kamada et al., 2013). Its precise molecular function, however, is not clear yet. To test whether ScpB is involved in the association of Smc–ScpA rings with chromosomes we combined the cysteine mutations in Smc and ScpA with an scpB in-frame deletion (Figure 4—figure supplement 1). Ring formation was only mildly affected by the absence of ScpB as judged by BMOE cross-linking and in-gel fluorescence detection (Figure 4A) (Bürmann et al., 2013). However, Smc complexes lacking ScpB subunits failed to entrap chromosomes altogether demonstrating that ScpB is absolutely required for loading of prokaryotic condensin onto chromosomal DNA.10.7554/eLife.06659.010Figure 4.ScpB and ParB are essential for efficient DNA entrapment by Smc complexes.

Bottom Line: Exclusively species of Smc-ScpA, which were previously cross-linked into covalent rings, remained associated with chromosomal DNA.DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted, implying that most Smc-ScpAB is loaded onto the chromosome at parS sites near the replication origin.We furthermore report a physical interaction between native Smc-ScpAB and chromosomal DNA fragments.

View Article: PubMed Central - PubMed

Affiliation: Chromosome Organization and Dynamics, Max Planck Institute of Biochemistry, Martinsried, Germany.

ABSTRACT
Smc-ScpAB forms elongated, annular structures that promote chromosome segregation, presumably by compacting and resolving sister DNA molecules. The mechanistic basis for its action, however, is only poorly understood. Here, we have established a physical assay to determine whether the binding of condensin to native chromosomes in Bacillus subtilis involves entrapment of DNA by the Smc-ScpAB ring. To do so, we have chemically cross-linked the three ring interfaces in Smc-ScpAB and thereafter isolated intact chromosomes under protein denaturing conditions. Exclusively species of Smc-ScpA, which were previously cross-linked into covalent rings, remained associated with chromosomal DNA. DNA entrapment is abolished by mutations that interfere with the Smc ATPase cycle and strongly reduced when the recruitment factor ParB is deleted, implying that most Smc-ScpAB is loaded onto the chromosome at parS sites near the replication origin. We furthermore report a physical interaction between native Smc-ScpAB and chromosomal DNA fragments.

No MeSH data available.