FIG. 7.
Depletion of Sth1 severely reduced joining and processing of DSB at the MAT locus. (A) NHEJ proficiency was assayed by using qPCR with oligonucleotides that anneal to each side of DSB. Cultures were induced to express HO for 1 h and switched to the YEP-glucose medium that shuts off HO expression. The percent NHEJ efficiency was calculated by the equation (Tx − T0)/(Tun− T0) × 100, using the amount of PCR product prior to HO expression (Tun), immediately after 1 h of HO expression (T0), and during the recovery following HO expression (TX), all of which were normalized by the control (PRE-1) PCR. In this assay, almost every surviving cell religated and restored the complete MATα locus by precise end joining (25). Each point represents the average for three or more separate experiments. The standard deviation is shown. ChIP assays were used to assess the levels of RPA (b) or Rad51 (c) in the presence or absence of Sth1 at the DSB using an anti-RPA antibody (a gift from S. Brill) or anti-Rad51-antibody (a gift from P. Sung) and the level of Rad51 at the HML donor (d). Purified DNA was analyzed by qPCR using multiple sets of primers. See the legend to Fig. 4 for the definition of IP.