Essential role of MCM proteins in premeiotic DNA replication

Abstract

A critical event in eukaryotic DNA replication involves association of minichromosome maintenance (MCM2-7) proteins with origins, to form prereplicative complexes (pre-RCs) that are competent for initiation. The ability of mutants defective in MCM2-7 function to complete meiosis had suggested that pre-RC components could be irrelevant to premeiotic S phase. We show here that MCM2-7 proteins bind to chromatin in fission yeast cells preparing for meiosis and during premeiotic S phase in a manner suggesting they in fact are required for DNA replication in the meiotic cycle. This is confirmed by analysis of a degron mcm4 mutant, which cannot carry out premeiotic DNA replication. Later in meiosis, Mcm4 chromatin association is blocked between meiotic nuclear divisions, presumably accounting for the absence of a second round of DNA replication. Together, these results emphasize similarity between replication mechanisms in mitotic and meiotic cell cycles.

Figure 1

P-factor arrests cells with chromatin-associated Mcm4. (A) Experimental procedure. (B) Chromatin binding assay showing Mcm4-GFP chromatin association (top panels) and phase/DAPI (bottom panels) at various times after P-factor addition. Bar: 10 μm. (C) Quantitation of chromatin binding assay, showing percentage of uninucleate and binucleate cells positive for Mcm4-chromatin binding. “- triton” shows percentage of cells with nuclear localization of Mcm4 when the Triton-extraction step is omitted. Error bars show the range of data from two or more experiments. (D) Flow cytometric analysis of cells for data points shown in B and C).

Figure 2

Cells arrested in G1 by cyclin B shut off have chromatin-associated Mcm4p. (A) Experimental procedure. (B) Chromatin binding assay showing Mcm4-GFP chromatin association (top panels) and phase/DAPI (bottom panels) at various times after addition of thiamine to the medium. Bar: 10 μm. (C) Quantitation of chromatin binding assay, showing percentage of uninucleate cells positive for Mcm4-chromatin binding with or without Triton extraction. (D) Flow cytometric analysis of cells for data points shown in B and C.

Figure 3

Mcm4p associates with chromatin during premeiotic S phase. (A) Experimental procedure. See text for details. (B) Chromatin binding assay on cells after induction of meiosis, showing Mcm4-GFP chromatin association (left panels) and phase/DAPI (right panels). Bar: 10 μm. (C) Quantitation of chromatin binding assay, showing percentage of cells positive for Mcm4-chromatin binding with or without Triton extraction. (D) Flow cytometric analysis of cells for data points shown in B and C.

Figure 4

Mcm4 remains nuclear and associates with chromatin during premeiotic S phase in diploid cells, but chromatin binding is blocked between meiosis I and II. Strain P978 was arrested in G1 and induced to enter meiosis as in Figure 3A, except that a temperature shift to 34°C was used. (A) Nuclear localization (-Triton) and chromatin binding (+Triton) of Mcm4-GFP was determined at times shown after the shift to 34°C. Right-hand images show phase/DAPI-staining. Cells were either fixed in methanol and acetone without extraction (-Triton), or detergent extracted using the chromatin binding assay (+Triton). Bar: 10 μm. Progress of meiosis was monitored by following nuclear divisions (B) and flow cytometry (C). (D) Quantitative analysis of data for experiment shown in A–C, indicating percentage of cells showing Mcm4 nuclear localization (-Triton) and chromatin association (+Triton) at different stages of meiosis. Note chromatin binding of Mcm4 during premeiotic S phase (top panel, 2 h) and absence of chromatin binding but maintenance of nuclear localization of Mcm4 between meiosis I and II (middle panel, 5–6 h). Increase in nuclear Mcm4 in extracted cells with 4 nuclei (bottom panel, 6–7 h) reflects formation of spores that are resistant to zymolyase digestion and thus detergent extraction. ND, not determined.

Figure 5

Inhibition of premeiotic S phase with HU prevents displacement of MCM2–7 proteins. (A) Experimental procedure. Strains used were P958 (Mcm4p), P1027 (Mcm2p), and P1025 (Mcm6p). (B) Chromatin binding assay on cells before induction of meiosis and after induction of premeiotic S phase, showing Mcm2, 4, and 6-GFP chromatin association (left panels) and phase/DAPI (right panels). Bar: 10 μm. Flow cytometric analysis of cells is shown underneath. (C) Mcm4 function is essential for Mcm2 chromatin binding. The experiment shown in Figure 2B was repeated using an Mcm2-GFP strain (P1096) containing a temperature-sensitive mcm4 mutation (mcm4ts-td, a degron allele). No chromatin association of Mcm2-GFP is seen after the temperature shift in the presence of hydroxyurea. (D) Quantitation of data shown in B and C.

Figure 6

Analysis of mcm4 degron mutant. (A) Flow cytometric analysis of degron mcm4 (mcm4ts-td, P1023) and mcm4ts (cdc21-M68, P1) strains during the vegetative cell cycle, after shifting asynchronous cultures to 37°C at t = 0, showing that the degron strain arrests with 1C DNA. (B) Mcm4 protein levels decline abruptly on shifting the mcm4 degron strain (mcm4ts-td, P1023) to the restrictive temperature. In contrast, Mcm4 levels in the mcm4ts (cdc21-M68, P1) strain are not affected by the temperature shift. α-Tubulin is shown as a loading control. (C) mcm4 degron mutation blocks premeiotic S phase. pat1ts mcm4ts-td (P1037) and mcm4⁺ pat1ts (P937) strains were arrested in G1 and then shifted to 37°C and re-fed as in Figure 4A, except that cells were shifted to 37°C 30 min before refeeding. Progress of premeiotic DNA replication was analyzed by flow cytometry. (D) Analysis of meiotic progress for experiment shown in C and E, showing that inactivation of Mcm4 inhibits nuclear divisions. (E) Mcm4 levels are reduced in pat1ts mcm4ts-td (P1037) mutant on nitrogen starvation and induction of meiosis at 37°C compared with a mcm4⁺ pat1ts (P937) strain (time after shift is shown). α-Tubulin is shown as a loading control.