(A) Sequence alignments of Mer2 orthologs from multiple clades revealed a previously undescribed conservation in the N-terminal region. We created two mutants, the ‘3A’ mutant (W58A, K61A, L64A; red stars) based on three universally conserved residues and the ‘4A’ mutant (D52A, E68A, R70A, E71A; yellow stars). The periodicity of the red star conserved residues, combined with some secondary structure predictions, suggest that these residues might be on the same face of an alpha-helix. (B) Western blot analysis of meiotic yeast cultures of wildtype, mer2Δ, MER2-3HA mer2Δ, mer2-3a-3HA mer2Δ, and mer2-4a-3HA mer2Δ strains. Time of induction into the meiotic program indicated. See Yeast strains used in this study for strain information. Pgk1 was used as a loading control. Please note the indicated phospho-Hop1, anti-phospho histone H3 was used to measure the activation of Mek1 kinase in response to DSB formation. (C) Quantification of spore viabilities of wildtype, mer2Δ, MER2-3HA mer2Δ, mer2-3a-3HA mer2Δ, and mer2-4a-3HA mer2Δ strains. Number of dissected tetrads is indicated. See Yeast strains used in this study for strain information. (D) Representative images of meiotic chromosome spreads stained for Mer2 (α-HA [green], Hop1, and DNA [blue]) using 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) from wildtype, MER2-3HA mer2Δ, mer2-3a-3HA mer2Δ, and mer2-4a-3HA mer2Δ strains at t = 3 hr after induction into the meiotic time course. (E) Quantification of the number of Mer2 foci on chromosome spreads of strains used in (D). Mean and standard deviation are indicated. *p ≤ 0.05, **p ≤ 0.01, and ****p ≤ 0.0001; n.s. (non-significant) >0.05. Mann-Whitney U test. Number of analysed cells is indicated. (F) Volcano plots of quantitative immunoprecipitation (IP) mass spectrometry of Mer2WT vs. Mer24A. Proteins are grouped into hits (red) and candidates (blue), according to the fold change and p-value. Proteins appearing on the left show reduced association with Mer23A when compared with Mer2WT, whereas proteins on the right show an increased association with Mer24A when compared with Mer2WT. Circled in yellow are the histone proteins Hht1 (histone H3) and Htb1/Htb2 (histone H2B). (G) Detail of Mer24A vs. Mer2WT in IP quantitative mass spectrometry analysis for Hht1 (histone H3); top panel, and Hop1; lower panel. Association with Hht1 is reduced ~2-fold in Mer24A whereas it is essentially unchanged for Hop1.