Figure 5.

Joint DNA molecules arise at similar time and frequency at the ade6-3101 and ade6-3049 DSB hotspots. mus81Δ strains were induced for meiosis, and DNA, digested with BsrGI, was analyzed by two-dimensional gel electrophoresis and Southern blot hybridization using a probe near the right end of the 11.8 kb fragment with ade6. (A) Branched DNA molecules, predominantly replication intermediates (Y-shaped; thick white arrows), arose at 2 h, and recombination intermediates (X-shaped Holliday junctions; thin black arrows) appeared at 4–6 h. The prominent spot is the parental DNA fragment. (B) Expanded view of replication arc at 2 h, showing a prominent pause or DSB site in the ade6-3101 strain (bottom panel) but not in the ade6-3049 strain (upper panel). (C) For quantification, branched DNA (structures above the linear DNA arc) was normalized to total DNA. Quantification is based on two or three blots from two independent inductions; error bars indicate the range or SEM.