Volume 10, Issue 2, Pages 373-385 (August 2002) Recovery from Checkpoint-Mediated Arrest after Repair of a Double-Strand Break Requires Srs2 Helicase  Moreshwar B Vaze, Achille Pellicioli, Sang Eun Lee, Grzegorz Ira, Giordano Liberi, Ayelet Arbel-Eden, Marco Foiani, James E Haber  Molecular Cell  Volume 10, Issue 2, Pages 373-385 (August 2002) DOI: 10.1016/S1097-2765(02)00593-2

Figure 1 Experimental System for Recovery (A) HO endonuclease cleavage sites (HOcs) at MAT, HML, and HMR were deleted, and an HOcs was inserted inside the LEU2 open reading frame (leu2-cs). A segment immediately proximal to the cut site was inserted approximately 25 kb away to generate strain YMV80. The DSB is repaired by single-strand annealing. Strains YMV2, YMV45, and YMV86 are very similar except that the distance between the DNA repeats is either 30, 5, or 0.7 kb, respectively. (B) Viability on YEP-galactose plates, where HO endonuclease is induced to create a DSB, repaired by SSA. Molecular Cell 2002 10, 373-385DOI: (10.1016/S1097-2765(02)00593-2)

Figure 2 Recovery from Checkpoint Arrest in Wild-Type Cells (A) Physical analysis of single-strand annealing. The identities of various bands are indicated. (B) FACS analysis. The peaks corresponding to 1c and 2c DNA content are indicated. (C) Phosphorylation of Rad53p on a Western blot (top panel). In situ autophosphorylation of Rad53p, as described in the Experimental Procedures (middle panel). Phosphorylation of Chk1 on a Western blot (bottom panel). Molecular Cell 2002 10, 373-385DOI: (10.1016/S1097-2765(02)00593-2)

Figure 3 A rad52 Deletion Prevents Repair, but the Cells Adapt (A) Physical analysis of HO cutting and repair. A Southern blot of DNA extracted at intervals after HO induction is shown. Note that there is no formation of product and that all leu2 segments are degraded. (B) FACS analysis. (C) Phosphorylation of Rad53p as shown by Western blot (top panel) and by autophosphorylation activity of Rad53p (bottom panel). Molecular Cell 2002 10, 373-385DOI: (10.1016/S1097-2765(02)00593-2)

Figure 4 cdc5-ad and yku70Δ Mutants Prevent Adaptation but Not Recovery (A) rad52Δ cdc5-ad and rad52Δ yku70Δ mutants remain arrested with Rad53p hyperphosphorylation (top) and elevated kinase activity (bottom). Shown are (B) FACS analysis and (C) phosphorylation and kinase activity of Rad53 in RAD52 cdc5-ad and RAD52 yku70Δ mutants, which can recover. Molecular Cell 2002 10, 373-385DOI: (10.1016/S1097-2765(02)00593-2)

Figure 5 srs2Δ Mutants Are Defective in Recovery (A) Southern blot analysis of SSA-mediated deletion formation. (B) srs2Δ mutants die as dumbbell-shaped cells, whereas wild-type cells recover between 8 and 16 hr. (C) Western blot showing Rad53 phosphorylation. Molecular Cell 2002 10, 373-385DOI: (10.1016/S1097-2765(02)00593-2)

Figure 6 Suppression of srs2Δ Recovery Defect (A) mec1Δ sml1Δ and rad51Δ mutations suppress the recovery defect of srs2Δ. Survival of the indicated strains on galactose plates is shown. (B) Checkpoint override by caffeine suppresses lethality of srs2Δ cells. Viability of YMV80 (circles) or YMV80 srs2Δ cells (triangles) without (open symbols) or with the addition of caffeine (filled symbols) is shown. (C) Lethality in srs2Δ mutants depends on the amount of ssDNA that can accumulate during single-strand annealing, which correlates with the proportion of cells that activate the DNA damage checkpoint and the length of G2/M arrest. (D) Rad53p phosphorylation in srs2Δ cells during an SSA event between two leu2 segments 5 kb apart, which takes less than 1.5 hr for completion. (E) srs2Δ cells produce product that is intact on both the strands. Physical analysis following alkaline agarose gel electrophoresis is shown. (F) Recovery is delayed in the rad51Δ mutant. Molecular Cell 2002 10, 373-385DOI: (10.1016/S1097-2765(02)00593-2)

Figure 7 srs2Δ Cells Exhibit a Recovery Defect Even during an Ectopic Gene Conversion Event (A) The experimental system for ectopic gene conversion. (B) FACS analysis. (C) Viability on YEP-galactose plates. (D) Southern blot analysis of the gene conversion (gc) event. Gene conversion restores the HO-cleaved restriction fragment that is nearly absent after 1 hr induction of the endonuclease. Gene conversion accompanied by crossing over (<10% of total products) generates two different-sized reciprocal crossover bands, one of which is well resolved from the noncrossover band below it. Molecular Cell 2002 10, 373-385DOI: (10.1016/S1097-2765(02)00593-2)