Figure 5.

Effect of mec1-85 mutation on association of Mec1 and Ddc2 with the HO-induced DSB. (A) Schematic of the HO cleavage site at the ADH4 locus (ADH4cs). An HO cleavage site, marked with HIS2, was introduced at the ADH4 locus on chromosome VII. The primer pairs were designed to amplify regions 1 and 2 kb apart from the HO cleavage site. An arrow represents telomere. (B) Effect of mec1-85 mutation on Mec1 association with HO-induced DSBs. Cells expressing Mec1-HA (KSC1635), Mec1-85-HA (KSC1646), or Mec1-KN-HA (KSC1645) were transformed with YCpA-GAL-HO plasmid. Transformed cells were initially grown in sucrose and arrested at G₂/M with nocodazole. The culture was then incubated with galactose to induce HO expression for 3 h, whereas part of the culture was maintained in sucrose to repress HO expression. Cells were collected and subjected to chromatin immunoprecipitation. PCR was carried out with the primers for the HO cleavage site at the ADH4 locus and for the control SMC2 locus (see A). PCR products from the respective input extracts are shown below. (C) Effect of mec1-85 mutation on Ddc2 association with HO-induced DSBs. Wild-type (KSC1717) and mec1-85 (KSC1647) cells expressing Ddc2-HA were transformed with YCpA-GAL-HO plasmid and were analyzed as in B.