Figure 6.
Interdependent association of Mec1 and Ddc2 with the HO-induced DSB. (A) Effect of ddc2Δ mutation on Mec1 association with HO-induced DSBs. Wild-type (KSC1635) and ddc2Δ (KSC1636) cells expressing Mec1-HA were analyzed as in Figure 5B. All the strains contained an sml1Δ mutation, which suppresses the lethality associated with ddc2Δ mutation (Wakayama et al., 2001). (B) Effect of mec1Δ mutation on Ddc2 association with HO-induced DSBs. Wild-type (KSC1717) and mec1Δ (KSC1727) cells expressing Ddc2-HA were analyzed as in A. (C) Intracellular localization of Ddc2 in wild-type and mec1Δ cells. Wild-type (KSC1304) and mec1Δ (KSC1302) cells expressing Ddc2-myc were harvested and spheroplasted. Spheroplasts were homogenized to prepare whole cell extract (W) and then separated into the cytoplasmic (C) and nuclear (N) fractions. Aliquots were analyzed on immunoblots with anti-HA, anti-glucose-6-P dehydrogenase (G6PDH), and anti-nuclear pore complex (NPC) antibodies. (D) Top, dosage effect on Ddc2 association with HO-induced DSBs. Wild-type (KSC1717) and mec1Δ (KSC1727) cells expressing Ddc2-HA were transformed with YCp-DDC2-HA or the control vector and were analyzed as in A. Bottom, to monitor the expression level of Ddc2-HA, the extract was also analyzed by immunoblotting analysis.