Figure 4.

Isw2 and Ino80 facilitate efficient firing of rDNA origin of replication. (A) Schematic drawing of 2D gel with features annotated. The 1N spot is comprised of restriction fragments that are not in the process of replicating; the Y arc of restriction fragments that are being passively replicated; and the bubble arc of restriction fragments in which an origin of replication has actively fired. Replication fork pausing at the RFB causes an accumulation of restriction fragments with a specific size and shape, visible as a dark spot on the left arm of the Y-arc. The ratio of bubble arc to Y arc signal is indicative of the ratio of actively to passively replicated restriction fragments, and thus of origin efficiency. (B) Representative 2D gels of NheI-digested DNA over rARS and RFB, from cells grown in YPD at 30°. Exposures of the blots have been adjusted so that the Y arc is of comparable intensity for each blot, such that direct comparison of bubble arc intensity across images is equivalent to a comparison of bubble-to-Y ratio. Bubble arc indicated by empty arrow, Y arc indicated by filled arrow. Quantification based on measurement of average intensity of arcs using ImageQuantTL software, and reflects at least two independent experiments for each genotype. All values normalized to the bubble:Y ratio for wild-type. Error bars show SEM. Statistical significance determined by pairwise t-tests followed by Bonferroni correction for multiple testing. * P < 0.05. (C) As in (B), but from cells grown in YPD at 23°. Statistical significance determined by Welch’s t-test. * P < 0.05.