Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast

Abstract

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.

Figure 1. DSB formation is reduced by the tel1 mutation and the effect is chromosome specific.

Diploid sae2, sae2 pch2 and sae2 tel1 mutants in the NDT80 positive background or the ndt80 mutant background were introduced into meiosis and DSB formation was detected at indicated time points in chromosomes VII (A) and II (B). Lane profiles of 10 and 12 hours in each mutant background were normalized and averaged to obtain the profiles shown on the right. Cells from the same time course were used to examine both chromosomes VII and II. The Southern blot data used for sae2 and sae2 pch2 are the same as previously shown in .

Figure 2. Quantitative analysis of meiotic DSB formation.

DSB numbers were calculated using Southern blot data and the formula described in Materials and Methods. (A) The effect of the tel1 mutation on DSB formation. (B) The tel1 mutation reduces DSB formation in the absence of Ndt80 in chromosome VII and II. (C) rad17-mn effect on DSB formation in the presence of various mutations. A whole chromosome was used for DSB number calculation in the sae2 mutant strains while one third of a chromosome was employed in the rad51 dmc1 mutant strains (Materials and Methods). Error bars represent standard error. *, statistically significant (p<0.05, unpaired t-test). The actual data used to calculate DSB numbers are shown in Table S2.

Figure 3. Positive and negative effect of the rad17-mutation on DSB formation.

Diploid rad51 dmc1 strains carrying various mutations as indicated, in the NDT80 positive background or the ndt80 mutant background, were introduced into meiosis and DSB formation was detected at indicated time points in chromosomes VII (A) and II (B).

Figure 4. Comparison of lane profiles of broken meiotic chromosomes.

Lane profiles of Southern blot signals shown in Figure 3 were compared between various mutants as indicated. Lane profiles of 10 and 12 hours in each mutant background were normalized and averaged to obtain the profiles shown.