Figure 1. Single-Strand DNA Formation, γ-H2AX Formation, and Mre11p/Rad51p Binding in Response to a DSB at the MAT Locus.
HO cutting at MAT was induced either in asynchronously growing cells (“cycling,” H1069) or in cells arrested by exposure to α-mating pheromone (“G1,” H1072), and samples were collected at the appropriate time points (see Experimental Procedures).
(A) Map of the region immediately centromere-distal to the MAT DSB site. The map shows the method used to detect DSB formation and 5′-to-3′ resection. DNA was digested with SspI and separated on alkaline agarose gels, and gel blots were hybridized with a single-strand probe specific to the unresected strand (ss probe). HO-cut and uncut chromosomes produce 0.9 kb and 1.2 kb fragments, respectively; 5′-to-3′ resection past SspI sites eliminates cutting at these sites and thus produces larger SspI fragments detected by the probe.
(B) Southern blot illustrating this analysis. Bands labeled r1 through r6 are the products of resection through SspI sites 0.9, 1.6, 3.5, 4.7, 5.9, and 6.5 kb from the HO-cut site; * denotes a cross-hybridizing band that was used as a loading control.
(C) Quantitative analysis of the blot in (B) and of one replicate. (Filled squares, filled circles) Percent of chromosomes with DSBs; (open squares, open circles) percent of DSB-containing chromosomes that have had at least 0.9 kb resected. (filled squares, open squares) Data from cycling cells; (filled circles, open circles) data from G1-arrested cultures.
(D) Examples of multiplex PCR products of ChIP reactions used to determine relative amounts of γ-H2AX formed and of Mre11p bound near the MAT DSB. All reactions contained primer pairs for a sequence 66 kb from the break (“con”) and experimental sequences (“dsb”) 5.1 kb to the left for γ-H2AX and 0.02 kb to the right for Mre11p. (+) ChIP prepared with the indicated primary antibody; (−) treated identically but without primary antibody; (DNA) PCR reactions for which genomic DNA was used.
(E) Timing of γ-H2AX formation (filled squares, open squares) and Mre11p binding (filled circles, open circles) in cycling (filled symbols) and G1-arrested (open symbols) cultures. Data are from panels in (D) and 3–4 replicate experiments. For normalizing DSB-specific relative ChIP values (dsb/control band intensity ratios, see Supplemental Experimental Procedures), the maximum value obtained for each time course was set to unity. Symbols and error bars report the average and standard deviation for these normalized values.