(A–C) Meiotic prophase. Nuclear spreads from meiotic MER2myc cultures were stained with DAPI (grayscale), anti-myc antibodies (green), and anti-Zip1 antibodies (red). Equivalent exposures of representative nuclei at the indicated stages are presented.
(D, E) Premeiotic nuclei. Spreads from MER2myc (D) and untagged MER2 (E) strains were stained with DAPI (grayscale) and anti-myc (green). Equivalent exposures are presented.
(F, G) Early meiotic nuclei. Spreads from a MER2myc strain were prepared two hr after transfer to sporulation medium, and stained with DAPI (grayscale), anti-myc (green), and anti-Red1 (red). All scale bars represent 1 μm.

(A) Mer2myc expression. Denaturing whole-cell extracts were prepared from untagged diploid cells or MER2myc haploid (MATa) or diploid cells in log phase culture (cycling cells) or at the indicated times after transfer to sporulation medium (cells in SPM). Extracts were analyzed by SDS-PAGE and western blotting with anti-myc antibody. The asterisks in (A) and (D) indicate a non-specific band frequently seen with cells subjected to the standard meiotic pre-growth regimen. The blot was stripped and reprobed with anti-tubulin as a loading control.
(B) Time course of Mer2myc expression in meiosis. Denaturing extracts were prepared at the indicated times after transfer to sporulation medium and analyzed by anti-myc western blotting. A lighter exposure is shown than the blot in (A) to reveal multiple shifted forms. Meiotic timing of the same culture was determined by measuring SC formation and nuclear division.
(C) Mer2myc phosphorylation. Denaturing whole-cell extract was prepared at 4 hr in meiosis, then Mer2myc was immunoprecipitated and treated with lambda phosphatase alone or in the presence of inhibitors.
(D) Mer2myc phosphorylation in DSB-defective mutants. Denaturing extracts from the indicated mutants at 4 hr in meiosis were analyzed by anti-myc western blotting.

(A) Western blot of whole-cell extracts from cdc28-as1 meiotic time courses. The culture was divided and either left untreated or treated starting at 0 hr with 0.5 or 5 μM 1-NM-PP1. The asterisk denotes a Mer2myc proteolytic product. Half as much material was loaded for the drug-treated samples.
(B) Western blot of whole-cell extracts from a clb5δ clb6 δ strain.
(C) Direct phosphorylation of Mer2 by Cdc28 in vitro. Kinase assays with [γ³²P] N⁶-(benzyl)ATP were performed with whole-cell extract prepared at 3 hr in meiosis from CDC28 (lane 1) and cdc28-as1 cultures (lanes 2–7). Extracts were incubated without added substrate (lane 2), with histone H1 (lanes 1 and 3), or with the indicated recombinant Mer2 proteins purified from E. coli. The upper panel is an autoradiograph; the lower panel shows the relevant portion of the same gel stained with Coomassie Blue. Dashes indicate positions of full-length Mer2 and a proteolytic fragment present in the recombinant protein preparation.

(A) Two Cdc28 consensus sites in Mer2 (underlined). Black boxes indicate residues identical to those in S. cerevisiae. N-terminal sequences were not available for S. kluyveri.
(B, C) Western blot analysis of meiotic time courses of wild-type and Cdc28-consensus-site mutants (MER2myc, mer2(S30A)myc, mer2(S271A)myc, and mer2(S30,271A)myc). In panel C, samples from 4 hr and 6 hr time points are compared side by side; half as much extract was loaded for the mer2 mutants. Blots were stripped and reprobed for tubulin as a loading control.
(D) Residual phosphorylation of Mer2(S30A) and Mer2(S30,271A). Myc-tagged protein was immunoprecipitated from denaturing whole-cell extracts prepared at 6 hr in meiosis, and left untreated or treated with lambda phosphatase.
(E) Spore viabilities (100 tetrads per strain).
(F) Intragenic meiotic recombination at the his4LEU2 hotspot in return-to-growth assays (untagged MER2 alleles). Each value is the mean ± s.d. for three independent cultures.
(G) DSBs at the YCR048w hotspot, measured by Southern blotting of genomic DNA from rad50S strains (untagged MER2 alleles).
(H) Association of Mer2myc with meiotic chromatin. The subcellular fractionation assay is outlined at the top. Extracts from cells at 4 hr in meiosis were fractionated and analyzed by western blotting. Lanes contain equal cell equivalents. Asterisks denote a Mer2myc proteolytic product.

(A) Schematic of the Mer2 interactions analyzed.
(B–F) Yeast two-hybrid analysis of wild-type and mutant Mer2 proteins. β–galactosidase (β–gal) reporter expression (mean ± s.d. for three independent cultures) is given for LexA and Gal4 activating domain (AD) fusions in the indicated combinations. In (D), “Mer2*” indicates that LexA was fused to the same version of Mer2 (wild type or mutant) as Gal4AD. Minus signs denote empty vector controls.

(A) Model for establishment of Mer2 phosphorylation patterns and interactions with other DSB proteins. See text for details.
(B) CDK directly promotes both premeiotic DNA replication and DSB formation.
(C) Fluctuation of B-type cyclin-Cdc28 kinase activities (based on Grandin and Reed, 1993; Stuart and Wittenberg, 1998). DSB formation is restricted to a window (shaded green) at the beginning of meiotic prophase. In this window, Clb5-Cdc28 activity is at intermediate levels but other cyclin-CDK activities are relatively low. We propose that a threshold of Clb5-Cdc28 activity is required to establish conditions permissive for DSB formation, and that this contributes to the appropriate timing of recombination initiation. Although DSBs form after DNA replication is completed locally, asynchrony of replication of different parts of the genome may result in formation of DSBs in early replicating regions before later regions have been replicated. The time (horizontal) axis is not drawn to scale.