Extending comparisons of in vivo and in vitro ORC-origin interaction strengths genome-wide.

<p>(A) Outline of genomic EMSA: Purified ORC at the indicated concentrations was incubated with 0.5 pM of purified sheared yeast genomic DNA. After the reactions reached equilibrium, ORC-bound and -unbound DNA fragments were separated by native gel electrophoresis. To mark the position of ORC-DNA complexes in the gel, a control reaction was performed with a specific radiolabeled origin-containing fragment (184 bp marker). (B) ORC binding in the gEMSA was visualized in MochiView <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003798#pgen.1003798-Homann1" target="_blank">[88]</a>. Screen shots are shown for two origins that were also examined by origin-specific EMSAs in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003798#pgen-1003798-g001" target="_blank">Figure 1</a>. The signals for each feature (oligo) on the array are indicated on the y-axis and the chromosomal coordinates are indicated on the x-axis. A magenta bar marks the coordinates comprising the oriDB annotated ARS. The signal strength for the 0.3 nM (green), 3 nM (blue) and 30 nM (orange) ORC concentrations are plotted and the coordinates corresponding to the ChIPotle called peaks at each concentration are shown by the correspondingly colored bars. The ORC-<i>ARS422</i> complex had an apparent Kd of 10 nM by origin-specific EMSAs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003798#pgen-1003798-g001" target="_blank">Figure 1E</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003798#pgen-1003798-t001" target="_blank">Table 1</a>), while the ORC-<i>ARS822</i> complex had an apparent Kd estimated to be >100 nM. (C) Heat maps depicting the ORC-origin binding strengths in the gEMSA for the 39 origins examined in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003798#pgen-1003798-g001" target="_blank">Figure 1E</a> or their normalized Kds (narrow map, center) are shown. gEMSA ORC-origin binding strength was defined two different ways, depending on the heat map. The heat map to the left (gEMSA peaks) defined the ORC-origin complex as the area of the peak that was called by the ChIPOTle program analysis of the gEMSA data in each independent array (ORC concentration shown at top of columns). If no binding peak was called, the ORC-origin binding strength was assigned a “0” value and colored white. The heat map on the right (<i>ORC2</i> ChIP coordinates) defined the ORC-origin complex in the gEMSA as the coordinates that comprised the ChIPOTle called ORC-origin peak for the previously published <i>ORC2</i> ChIP array <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003798#pgen.1003798-Shor1" target="_blank">[51]</a>. Thus in this map, DNA regions that were depleted in the experimental sample relative to total DNA were assigned a negative value and colored beige. The two different approaches produced similar conclusions, suggesting that for most of these origins the gEMSA peak corresponded well to the in vivo ORC-origin peak. (D) Correlation analysis of gEMSA-derived binding strength versus normalized apparent Kds as determined by origin-specific EMSAs in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003798#pgen-1003798-g001" target="_blank">Figure 1E</a>. The normalized apparent Kd values were calculated by dividing apparent Kds by the apparent Kd for <i>ARS317</i> (<i>HMR</i>-E). The gEMSA-derived binding strength was a quantitative expression of the data shown in the right map in © as described above. Specifically, the gEMSA signal for each ORC concentration for each origin was summed to give a “Total gEMSA signal.” Thus this value simply expressed a value for total ORC-origin complex formation observed across the ORC titration. Our analysis included 35 origins for which apparent Kd values could be determined. A Spearman correlation coefficient and P-value are indicated. (E) We grouped origins into classes based on their EMSA derived apparent Kd values and their sensitivity to the <i>orc2-1</i> mutation for ORC binding in vivo. We compared the Total gEMSA signal for each of these three groups: weak <i>orc2-1</i>s (apparent Kd>5x <i>ARS317</i>, n = 13), weak <i>orc2-1</i>r (apparent Kd>10x <i>ARS317</i>, n = 5), and tight <i>orc2-1</i>r (apparent KdARS317, n = 12). T tests were used to determine the significance of the difference between the weak or ‘chromatin-dependent’ groups and the DNA-dependent group. P-values are indicated in figure.