Function of Red1 interaction with the 9-1-1 complex

To study the functional significance of the observed Red1 interaction with 9-1- 1 during meiosis, we expressed the Red1 variants defective in either Mec3 binding (Q537_{A, V}540_{A; termed Red1}-Mec3_{) or Ddc1 binding (I}743_{A; termed Red1}- Ddc1

) as the only source of Red1 from the diploid genome. First, we tested spore survival of cells having the mutated form of Red1 as the only source in the diploid genome. Surprisingly, we detected a very mild phenotype in red1- Mec3 _{strains while the red1}-Ddc1 _{single or red1}-Mec3-Ddc1 _{double mutant led to} severely defective spores (Fig. 17A). These defects are in the range of the phenotypes observed in 9-1-1 deletions (Fig. 17A).

As a more specific assay and in order to test whether these red1 mutants are still capable in 9-1-1-dependent pachytene checkpoint signalling, we additionally deleted the gene for the meiotic recombinase Dmc1. Cells deficient in Dmc1 accumulate resected DSBs and recombination intermediates, which normally (when wild-type Red1 is expressed) activate the pachytene checkpoint (Bishop et al., 1992; Sacher et al., 2006). When we assayed for checkpoint activation by monitoring histone H2A serine-129 phosphorylation (equivalent to mammalian $-H2AX) and Rad52 SUMOylation (Sacher et al., 2006), we found no significant defect with mutants expressing the Red1 variant deficient in Mec3 binding (red1-Mec3_{) (data not shown). By}

Results Red1 binds 9-1-1 for checkpoint activation and normal SC formation

contrast, red1-Ddc1 _{mutants completely fail to induce the pachytene checkpoint} (Fig. 17B) reminiscent of 9-1-1-deficient mutants (Lydall et al., 1996), indicating that interaction of Red1 with the Ddc1 subunit of 9-1-1 is essential for this activity.

Figure 17: Red1 mutants deficient in Ddc1 binding show phenotypes in checkpoint activation. A: Spore viabilities of mutants. WT cells (SK1 strain) were compared with GFP-Zip1-expressing cells that express Red1 WT or 9-1-1 binding- deficient Red1 variants (red1-Mec3_{, red1}-Ddc1_{, red1}-Mec3, -Ddc1_{). Also shown are spore}

viabilities of 9-1-1 mutants (!rad17, !mec3, !ddc1). Strains were released into sporulation in 1,5% potassium acetate solution for 3 days before tetrade dissection, and spore viability was scored on YPD plates after 3 days. Indicated are the percentages of viable spores and the total number of spores counted (brackets). B:

Ddc1-binding-deficient Red1 variant (Red1-Ddc1_{) reverts pachytene checkpoint arrest}

of dmc1 deletion strains. Extracts of synchronously sporulating cells were made at the indicated times and probed by Western analysis for Zip1, and Pgk1 expression, and in parallel for phosphorylated H2A (equivalent to mammalian $H2AX) and Rad52 SUMOylation as measures for checkpoint activation.

Pachytene arrested cells also accumulate SCs and thus SC proteins like Zip1 because they do not progress further in the meiotic cell cycle (Roeder and Bailis, 2000). In !dmc1 strains, Red1 expression is normally induced, but accumulates until at least 24 hours in sporulation media (Fig. 17B), while in wild-type cells, Red1 expression reaches a maximum at around 6 hours and is hardly detectable after 24 hours. The accumulation of Red1 probably reflects the arrested state due to an active pachytene checkpoint and is observed in a number of other background strains arresting at certain points during early meiosis I (Fig. 21). We further observed that Zip1 is similarly expressed like Red1 in !dmc1 strains. The accumulation of both Red1 and Zip1 in a !dmc1 background was abolished when we integrated the red1-Ddc1 _{mutant as the}

Results Red1 binds 9-1-1 for checkpoint activation and normal SC formation

only source of Red1 into !dmc1 !red1 strains (Fig. 17B and data not shown).

!dmc1 strains with Red1 mutated in the Mec3 binding domain show no severe defects in all phenotypes tested, except a very mild defects in spore viability and a slightly delayed onset of Rad52 SUMOylation (data not shown). Altogether, these phenotypes argue for a crucial role of the Red1-Ddc1 interaction in pachytene checkpoint signalling.

Because the red1-Ddc1 _{mutation has a very strong effect on spore} viability (Figure 17A) and since Red1 is a structural component of the SC, we speculated that the mutant might also show deficiencies in SC formation. Indeed, when we assayed for SC formation utilizing GFP-tagged Zip1, we noticed a moderate defect in red1-Mec3 _{mutants, but a virtually complete loss of} normal SCs in red1-Ddc1 _{mutants and red1 mutants defective in interaction with} both 9-1-1 subunits (red1-Mec3,-Ddc1_{) (Fig. 18A). Interestingly, although pre-} assemblies of SCs were detectable, fully formed SCs were not formed if Red1 fails to bind 9-1-1 (Ddc1). Notably, the degree of SC formation defects of these red1 mutants was mirrored by their deficiencies in spore viability (Fig. 17A). The expression profile of Red1-Ddc1 _{during the meiotic time-course still}

shows a similar induction and decrease as in wild-type cells (Fig. 18B). The slightly reduced overall expression of mutated Red1-Ddc1

protein results from the inability to form mature SCs, but is not the reason for the observed phenotype in SC formation as cell with several Red1-Ddc1

integrations and therefore higher expression levels still show the very same defect in both pachytene checkpoint activation and SC maturation (data not shown). In contrast, red1-Mec3 _{mutants (and the red1}-Mec3,-Ddc1 _{mutant) do show changes in} the profile which can however not be connected to significant phenotypes in spore viability or SC formation assays (Fig. 17A; 18A,B). It is interesting to speculate that the unique profile of Red1 expression is triggered by its binding to the Mec3 subunit of 9-1-1. Moreover, Red1 expression seems to have direct influence on the levels of the central element protein Zip1 suggesting that Zip1 is stabilized by its interaction with Red1 (Fig. 18B).

Results Red1 binds 9-1-1 for checkpoint activation and normal SC formation

Figure 18: Red1 mutants deficient in 9-1-1 binding show phenotypes in SC formation. A: SC formation in WT and red1 mutants (9-1-1-binding mutants). SCs were visualized using GFP-tagged Zip1 and spinning disk microscopy as in Fig. 14C. Maturation of SCs was categorized in the indicated classes (early stage, diffuse, dot- like, pre-SCs and full SCs). In the quantified assay only pre-SCs and full SCs were distinguished. Cells were released into synchronous sporulation and samples observed after the indicated times. For each time-point more than 100 cells were analyzed. B: Red1 expression levels (from the identical experiment as in A).

Results Regulation of Red1 SUMOylation and expression