Figure 2.

MEK1 influences the repair of VDE–DSB1. (A) Restriction endonclease digestion with SpeI and probing a Southern blot as indicated (Pr) produces three bands. An 11.5-kb band containing DNA from three sources; parental arg4-vde (P), gene conversion products from using the arg4-bgl allele on the homologous template as donor (GC), parental arg4-bgl allele from the donor homologue (D). Two other bands are present, one at 7.8 kb contains the broken arg4-bgl alleles (VDE–DSB1) and one at 2.3 kb contains the deletion product of SSA (SSAΔ). Thick black line represents plasmid DNA, stippled line represents a natural Ty element. (B–D) Representative Southern blots with quantification from strains homozygous for the indicated MEK1 alleles. The graphs show duplicate experiments. The proportion of arg4-vde chromatids with a VDE–DSB1 (circles) or repaired to SSAΔ (squares) are calculated and plotted as described in ‘Materials and Methods’ section. The proportion of DNA in the P+GC+D band that is attributable to only P+GC was calculated as described and displayed for comparison (triangles). Removing Mek1 function by either deletion or using a site-specific mutation in the kinase domain increases the proportion of VDE–DSB1s repaired by SSA. By 8 h when almost all arg4-vde alleles have been cut (Figure 1B) and there is almost no unrepaired VDE–DSB1 detected, the proportion of repair by GC can be deduced (triangle at 8 h). GC is significantly reduced when Mek1 function is absent.