A checkpoint control linking meiotic S phase and recombination initiation in fission yeast

Abstract

During meiosis, high levels of recombination initiated by DNA double-strand breaks (DSBs) occur only after DNA replication. However, how DSB formation is coupled to DNA replication is unknown. We examined several DNA replication proteins for a role in this coupling in Schizosaccharomyces pombe, and we show that ribonucleotide reductase, the rate-limiting enzyme of deoxyribonucleotide synthesis and the target of the DNA synthesis inhibitor hydroxyurea (HU) is indirectly required for DSB formation linked to DNA replication. However, in cells in which the function of the DNA-replication-checkpoint proteins Rad1p, Rad3p, Rad9p, Rad17p, Rad26p, Hus1p, or Cds1p was compromised, DSB formation occurred at similar frequencies in the absence or presence of HU. The DSBs in the HU-treated mutant cells occurred at normal sites and were associated with recombination. In addition, Cdc2p is apparently not involved in this process. We propose that the sequence of meiotic S phase and initiation of recombination is coordinated by DNA-replication-checkpoint proteins.

Fig. 1.

RNR is required for DSB formation. (A) Position of the probe corresponding to the telomere-proximal portion of the NotI J fragment of chromosome 1. (B) pat1 (HM1307) and pat1 cdc22 (HM1936) cells were grown and then transferred to nitrogen-free medium for 16 h at 24°C to arrest cells in G₁ phase. Meiosis was induced by shifting the temperature to 36°C at time 0. Samples were subjected to digestion of DNA with NotI and then analyzed by PFGE and Southern blot hybridization with the probe described above. (C and D) pat1 (HM1307) (C) and pat1 rad50S (HM1691) (D) cells were arrested in G₁ phase, and meiosis was induced at 34°C in the absence (–) or presence (+) of 24 mM HU. Samples collected at the indicated times were analyzed as in B.(E) Quantitation of meiotic DSB frequency at mbs1 and mbs2 determined from experiments similar to that shown in D. Data are means ± SE of values from three independent experiments.

Fig. 2.

HU treatment inhibits DSB formation by acting in meiotic S phase. (A) pat1 rad50S (HM3696) cells were arrested in G₁ phase, and meiosis was induced in the absence (–) or presence (+) of HU, with the exception that HU was added at the indicated times after the shift to 34°C. Samples were subjected to PFGE, as described for Fig. 1B. The probe used was the one as shown in Fig. 1A.(B) DNA content of the cells in A was determined by flow cytometry. (C) Quantitation of meiotic DSB frequency at mbs1 and mbs2 determined from experiments similar to that shown in A. Data are means ± SE of values from four independent experiments.

Fig. 3.

Rad3p is required for coupling of meiotic DNA replication to DSB formation. (A–D) pat1 rad3 (HM2877) (A), pat1 rad3 rec12 (HM2849) (B), pat1 rad3 rad50S (HM2690) (C), or pat1 rad3 rec12 rad50S (HM2699) (D) cells were arrested in G₁ phase, and meiosis was induced at 34°C in the absence (–) or presence of HU (+), with HU added at time 0. Samples collected at the indicated times thereafter were analyzed as described for Fig. 1B. The same probe was used as in Fig. 1 A. (E) Quantitation of meiotic DSB frequency at mbs1 and mbs2 for pat1 rad3 rad50S (HM2690) cells determined from experiments similar to that shown in C. Data are means ± SE of values from three independent experiments.

Fig. 4.

The DNA-replication-checkpoint proteins are required for coupling of meiotic DNA replication to DSB formation. (A) pat1 (HM34), pat1 rad1 (HM1482), pat1 rad9 (HM713), pat1 rad17 (HM731), pat1 rad26 (HM783), and pat1 hus1 (HM798) cells were arrested in G₁ phase, and meiosis was induced at 34°C in the absence (–) or presence (+) of HU, with HU added at time 0. Samples collected at the indicated times thereafter were analyzed by PFGE, followed by ethidium bromide staining. Ch1, Ch2, and Ch3 indicated the positions of chromosomes 1 (5.7 Mb), 2 (4.6 Mb), and 3 (3.5 Mb), respectively. The smear (*) shows DSBs generated during meiosis. (B) pat1 cds1 rad50S (HM1801) cells were arrested in G₁ phase, and meiosis was induced at 34°C. Samples were analyzed as described for Fig. 1B. The probe used in B was the one shown in Fig. 1 A.

Fig. 5.

Cdc2p/Cdc13p is not involved in coupling meiotic DNA replication to DSB formation. (A and B) pat1 rad3 cdc2–22 (HM4065) (A) and pat1 cdc2–22 (HM4066) (B) cells were arrested in G₁ phase, and meiosis was induced at 36.5°C in the absence (–) or presence (+) of HU, with HU added at time 0. Samples collected at the indicated times thereafter were analyzed by PFGE as in described for Fig. 1B. The probe used was the one shown in Fig. 1 A.(C and D) pat1 rad3 cdc13 rad50S (HM4088) (C) and pat1 cdc13 rad50S (HM4090) (D) cells were arrested in G₁ phase, and meiosis was induced at 34°C in the absence or presence of HU, with HU added at time 0. Thiamine was added at time 0 to switch off cdc13⁺ expression. Samples collected at the indicated times thereafter were analyzed by PFGE and Southern blotting, as described above.