<i>SIR2</i> inhibited MCM association at the majority of euchromatic origins in <i>cdc6-4</i> mutant cells.

<p><b>A.</b> Experimental outline: MCM binding to chromosomal DNA was examined by ChIP-Seq from congenic <i>CDC6 SIR2</i> (wild type), <i>CDC6 sir2Δ</i> (<i>sir2Δ</i>), <i>cdc6-4 SIR2</i> (<i>cdc6-4</i>), and <i>cdc6-4 sir2Δ</i> cells. Cells were released from a G2/M nocodazole-arrest into G1 phase at 37°C prior to formaldehyde crosslinking. <b>B.</b> MCM ChIP-Seq signals across Chromosome III. The x-axis indicates the chromosomal coordinates, the y-axis the normalized MCM read counts (MCM signal). The scale on the y-axis is the same for each of the four strains. Confirmed origins on Chromosome III are indicated by boxes and numbers. Black boxes indicate origins that were defined as <i>SIR2</i>-responsive because they provided for plasmid replication in <i>cdc6-4 sir2Δ</i> cells at the non-permissive temperature for <i>cdc6-4</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref023" target="_blank">23</a>]. The arrowhead and the line-with-circle indicate <i>ARS305</i> and <i>ARS307</i>, respectively that produced similar association with MCM in wild type cells but different degrees of MCM association in <i>cdc6-4 sir2Δ</i> cells. These origins are highlighted to illustrate that the wild type pattern of MCM origin distribution was not rescued fully in <i>cdc6-4 sir2Δ</i> cells. The starred origins (<i>ARS301</i>, <i>ARS302</i>, <i>ARS317</i> and <i>ARS318</i>) are associated with transcriptional silencers that direct the assembly of <i>SIR</i>-heterochromatin at the <i>HML</i> and <i>HMR</i> loci on chromosome III [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref015" target="_blank">15</a>]. They were excluded from further analyses in this study for this reason, as discussed in text. <b>C.</b> MCM ChIP-Seq signals across Chromosome VI. The arrowhead and the line-with-circle indicate <i>ARS606</i> and <i>ARS609</i>, respectively, which behaved analogously to <i>ARS305</i> and <i>ARS307</i>, respectively, as described in B. <b>D.</b> Origins’ <i>SIR2</i>-responsiveness was defined as the ratio of the MCM signal at the origin in <i>cdc6-4 sir2Δ</i> cells relative to <i>sir2Δ</i> cells. We confined analyses to confirmed origins that produced strong signals in both WT and <i>sir2Δ</i> cells as defined by inclusion among the top 400 chromosomal coordinates of enrichment (<b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.s002" target="_blank">S2 Fig</a></b>). The dotted line indicates the cut-off that captured the euchromatic origins (<i>ARS305</i>, <i>ARS315</i>, <i>ARS603 and</i> ARS606) that had previously been defined as <i>SIR2</i>-responsive origins based on a systematic plasmid-based screen of these chromosomal origins [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007418#pgen.1007418.ref023" target="_blank">23</a>].