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.
Highlights
Homologous recombination is essential for the accurate segregation of homologous chromosomes during meiosis [1]
The efficiency of double-strand breaks (DSBs) formation was evaluated per chromosome using pulsed-field gel electrophoresis (PFGE) followed by Southern blotting, with probes recognizing an end of a particular chromosome [14]
We found that DSB formation was mildly affected in the absence of Tel1, with a more marked reduction in bigger chromosomes (IV, VII, II and XI) while DSB levels stayed similar in smaller chromosomes (VI and III) (Figure 1 and 2A)
Summary
Homologous recombination is essential for the accurate segregation of homologous chromosomes during meiosis [1]. The initiation of meiotic recombination is controlled by the genomewide formation of DNA double-strand breaks (DSBs) [2]. Meiotic DSBs are formed by the Spo protein, a meiosis-specific endonuclease that is homologous to type II topoisomerases [3]. Once Spo is removed from the 59 ends of DSBs, the 59 ends receive further resection, leading to the exposure of 39-ended single-stranded (ss) DNA strands [5]. These ssDNA strands are the substrates for homologous recombinases (i.e., RecA homologs, Rad and Dmc in yeast) that catalyze the homology searching and strand exchange reactions [6]. No meiotic DSBs are repaired in the absence of both Rad and Dmc1 [7]
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