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. 2018 Feb 14;14(2):e1007223. doi: 10.1371/journal.pgen.1007223

Fig 6. A mer2 point mutant affected for its interaction with Spp1 mimics the meiotic phenotype of spp1∆.

Fig 6

(A) Scheme of the Mer2 protein, with its predicted coil-coil structures (Hhpred) and the Spp1 interacting domain (165–232) [17]. Below is the Mer2 protein sequence in the predicted coiled-coiled heptad structure. Buried positions required for coiled-coil pairing are underlined. Amino acids conserved and predicted to be surface-exposed are colored. The mer2_sid mutation (V195D) is indicated by an arrow. (B) 2-hybrid assays for the interaction between Mer2 and Spp1. Growth on the–His medium indicates an interaction between the two tested proteins. (C) Association of Mer2 and Mer2_sid mutant proteins with chromosome axis during meiosis (t = 3 hr) monitored by ChIP. No tag: ORD7339; Mer2-Flag: VBD1251; Mer2_sid Flag: VBD1843. Each dot represents a biological replicate and the bar indicates the mean. (D) Interaction of Spp1 with Mer2 and the Set1 complex subunit Swd1 in meiotic cells examined by Western blot. Proteins pulled down by Spp1-TAP were released in the eluate after Tev cleavage of the Tap tag. Anti-HA, anti-Flag and anti-TAP antibodies were used as indicated. MER2-FLAG SPP1-TAP SWD1-HA: VBD1745; mer2_sid-FLAG SPP1-TAP SWD1-HA: VBD1852; SPP1-TAP SWD1-HA: VBD1742. (E) Association of Spp1 during meiosis (t = 3 hr) monitored by ChIP. No tag: ORD7339; WT: VBD1187; mer2∆: VBD1220; mer2_sid: VBD1924. Each dot represents a biological replicate and the bar indicates the mean. (F) Meiotic DSB formation monitored in dmc1∆ cells by Southern blot at CYS3 and DEP1 DSB (upper panel), or at the spp1∆-specific PES4 DSB. DSB sites are indicated by arrows. WT: ORD7354; mer2_sid: VBD1879; spp1∆: VBD1748. Graph shows the DSB quantification relative to the level in WT (for CYS3, DEP1) or spp1∆ cells (for PES4 site). DSB were quantified at the 5 hr time point. Each dot represents a biological replicate and the bar indicates the mean. (G) Meiotic progression as assessed by DAPI staining of strains with the indicated genotype. WT: ORD7339; mer2_sid: VBD1880; spp1∆: VBD1769; spo11Y135F: VBD1291; spo11Y135F spp1∆: VBD1233. Representative experiments are shown. See also S8 Fig.