Figure 5. A physical interaction of Smc–ScpAB rings and chromosomal DNA.

Co-purification of chromosomal DNA fragments with native Smc–ScpAB. Cells of strains BSG1104-5 and BSG1107-8 were treated with the cross-linker BMOE prior to cell lysis. Strains carrying (‘+’) or lacking (‘−’) cysteine mutations (‘6xCys’) in the Smc-AviTag construct were expressed in presence (‘+’) or absence (‘−’) of the biotin ligase (‘BirA’). Beads were washed in the presence of either a 150 mM ammonium acetate buffer (‘low salt’) or a 2 M sodium chloride buffer (‘high salt’). The co-purification of DNA fragments with Smc-biotin on streptavidin beads was measured by quantitative PCR using primer pairs specific for genomic positions indicated on a representation of the circular B. subtilis genome. Mean values and standard deviations were calculated from two independent biological replicates. The following figure supplement is available: Figure 5—figure supplement 1: Chromatin immuno-precipitation of Smc.

DOI: http://dx.doi.org/10.7554/eLife.06659.012

Figure 5—figure supplement 1. Chromatin immuno-precipitation of Smc.

All Smc variants used in Figure 5 behave similarly in standard ChIP experiments using anti-Smc antibodies. Cells of strains BSG1104-5 and 1107-8 were grown to mid-exponential phase and subjected to chromatin immunoprecipitation (ChIP) using polyclonal rabbit anti-Smc serum. Input and eluate DNA samples were analysed by quantitative PCR using specific primer pairs for the genomic positions indicated on a circular representation of the B. subtilis genome. Pull-down efficiency (ChIP-DNA/input-DNA *100%) was plotted for each primer pair. The lack and presence of the BirA biotin ligase or cysteines for cross-linking of Smc and ScpA proteins are denoted as ‘BirA−’ and ‘BirA+’ or ‘6xCys−’ and ‘6xCys+’, respectively. It is noted that the highly transcribed tRNA locus, trnS, is highly enriched by Smc-Avitag using standard ChIP (but not native ChIP, see Figure 5), being consistent with the notion that enrichment of highly transcribed genes by ChIP is prone to artefacts due to non-uniform formaldehyde cross-linking.

DOI: http://dx.doi.org/10.7554/eLife.06659.013