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.
Highlights
Compaction and individualization of sister DNA molecules is a prerequisite for efficient segregation of the genetic material to daughter cells during cell division
In Bacillus subtilis and Streptococcus pneumoniae, Smc–ScpAB is recruited to a region around the replication origin by ParB/Spo0J protein bound to parS sites, thereby forming a discrete focus— called condensation center—on each nascent copy of the chromosome (Gruber and Errington, 2009; Sullivan et al, 2009; Minnen et al, 2011)
We established an inverse assay by immobilizing whole chromosomes of B. subtilis in agarose plugs and monitoring their association with covalently closed rings of Smc–ScpA under harsh protein denaturing conditions (Figure 1A)
Summary
Compaction and individualization of sister DNA molecules is a prerequisite for efficient segregation of the genetic material to daughter cells during cell division. Inactivation of condensin subunits by mutation or depletion results in severe morphological aberrations and mechanical sensitivity of metaphase chromosomes, and subsequently to defects in their segregation during anaphase (Hirano and Mitchison, 1994; Ono et al, 2003; Gerlich et al, 2006). In Bacillus subtilis and Streptococcus pneumoniae, Smc–ScpAB is recruited to a region around the replication origin by ParB/Spo0J protein bound to parS sites, thereby forming a discrete focus— called condensation center—on each nascent copy of the chromosome (Gruber and Errington, 2009; Sullivan et al, 2009; Minnen et al, 2011). Inactivation of Smc–ScpAB in B. subtilis under nutrient rich
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