The authors have declared that no competing interests exist.
Conceived and designed the experiments: BA SF H. Tsubouchi. Performed the experiments: BA SF WL YT NH TT H. Tsubouchi. Analyzed the data: BA SF H. Tsubouchi. Contributed reagents/materials/analysis tools: BA SF H. Toyoizumi H. Tsubouchi. Wrote the paper: BA H. Tsubouchi.
Current address: School of Life and Medical Sciences, University College London, London, United Kingdom
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
Homologous recombination is essential for the accurate segregation of homologous chromosomes during meiosis
The mechanism for controlling meiosis-specific DSB formation has been extensively characterized using budding yeast as a model organism. Meiotic DSBs are formed by the Spo11 protein, a meiosis-specific endonuclease that is homologous to type II topoisomerases
Initiation of meiotic recombination needs to be coordinated with other events along the meiotic cell cycle. DSBs are most efficiently formed at the early stage of meiotic prophase I, and these DSBs finish being repaired toward the end of prophase I
Budding yeast has two major DNA damage checkpoint pathways that involve Mec1 and Tel1 respectively. Mec1 is the ortholog of ATR and is involved mainly in recognizing and responding to exposed ssDNA, whereas Tel1, the ortholog of ATM, responds to unprocessed DSB ends
With budding yeast as a model organism, we investigated the possible involvement of DNA damage checkpoint mechanisms in the regulation of meiotic DSB formation. We found that DSB formation was reduced in larger chromosomes in the
The
Diploid
DSB numbers were calculated using Southern blot data and the formula described in
Next we examined the possible involvement of the Mec1 pathway in DSB formation. We took advantage of the
The effect of the
Diploid
Lane profiles of Southern blot signals shown in
We previously showed that the
In mutants where DNA damage checkpoint mechanisms are compromised, defects can be a direct consequence of a failure to activate the DNA damage checkpoint or an indirect result of unscheduled cell cycle progression. To distinguish these possibilities, the
We investigated the possible roles of DNA damage checkpoint mechanisms in meiotic DSB formation using budding yeast. In order to quantitatively measure DSB formation, we employed genetic backgrounds in which DSB repair is defective; therefore the quantity of accumulated recombination intermediates is proportional to the amount of DSBs formed. However, the introduction of mutations in DNA damage checkpoint genes in such genetic backgrounds can cause a problem. The meiotic cell cycle in mutants defective in DSB repair is delayed/arrested in prophase I, and this phenomenon is suppressed when DNA damage checkpoint mechanisms are impaired. Since DSB formation usually occurs within prophase I, such unscheduled cell cycle progression itself can have a negative effect on DSB formation. By employing the
Tel1 is the ATM ortholog in budding yeast and primarily responds to unprocessed DSBs, such as those that persist in the
The recruitment of Tel1 to DSB sites depends on the Mre11-Rad50-Xrs2 (MRX) complex
In ATM deficient mice, the total level of Spo11-oligonucleotide complexes is elevated
Employing the
The difference in the usage of the ATM and ATR pathways in mice and budding yeast is interesting, given that ATM is primarily used for down-regulating DSB formation in mice. Once DSBs are formed, ATM is primarily used in responding to DSBs in mice whereas ATR (Mec1) is the major pathway in budding yeast. Thus, the apparent bias toward ATR utilization in budding yeast might reflect the overall usage preference to ATR in choosing a damage response pathway. After all, if meiotic DSBs, once formed, are processed to expose ssDNA in a relatively prompt manner, ATR would almost inevitably become the pathway of choice because ATM is less likely to be retained on processed DSB ends. On the other hand, in mice, it is possible that meiotic DSB ends are kept unprocessed for some time, which might allow ATM to respond to them, sending a negative feedback signal to the DSB forming mechanism.
Our results further highlighted the importance of Ndt80 as a negative controller of DSB formation. First, more DSBs are formed in the
Ndt80 is the master regulator that controls exit from prophase I and entry into metaphase I. Ndt80 is a downstream target of the DNA damage checkpoint mechanism during meiosis (recombination/pachytene checkpoint), which functions to coordinate homologous recombination (DSB repair) and cell cycle progression
The presence of unrepaired DSBs is sensed by DNA damage checkpoint mechanisms. In this work, we showed that Rad17 (and most likely the ATR pathway) is in charge of repressing DSB formation once DSBs are formed. Our results also suggest that DSB formation is shut off when cells exit prophase I. However, cells exit prophase I when the previously formed DSBs are repaired. This is contradictory because, as DSBs are repaired, the ATR-pathway becomes less active, possibly leading to reactivation of DSB formation. It is therefore likely that an unknown mechanism is responsible for gradually diminishing the DSB formation activity towards the end of prophase I. This mechanism may utilize the progress of homologous recombination as a temporal marker for prophase I. For example, the loading of proteins involved in later stages of homologous recombination, such as resolvases, and the formation of the synaptonemal complex can be exploited to serve such roles.
Genotypes of yeast strains are given in
Strains used are: TBR5514, 5515, 5188, 6618, 6619 and 6620 in
SK1 strains were introduced into meiosis as described previously with minor modifications
Meiotic DSBs were detected as described previously with minor modifications
Calculations to obtain the estimated DSBs on chromosomes used in
Based on this equation, E[N] solely relies on the signal ratio of unbroken chromosomes per total lane signal. This is an accurate estimate based on the assumption that 100% of cells enter into meiosis and the overall DSB distribution is not substantially affected by the introduced mutations. However, when the ratio of unbroken chromosomes becomes very small (<5%), the calculation is more easily affected from other factors such as a fraction of cells that did not go into meiosis and the quality of Southern blot. Thus, in the
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We would like to thank Angelika Amon, Neil Hunter and Matt Neale for strains.