On 2017 Jun 09, GERALD SMITH commented:

In Ma et al. (1), we isolated Schizosaccharomyces pombe ctp1 point mutations, which we interpreted to separate genetically two known meiotic activities that depend on Ctp1 for meiotic DNA break repair. A later paper by Jensen and Russell (2) claimed these activities are not genetically separable. We believe the disparity in interpretation stems largely from the use of meiotic cells in our studies but mitotic cells by Jensen and Russell. We maintain that in meiotic cells the activities are genetically separable.

During S. pombe meiosis, Rec12 (Spo11 homolog) makes DNA double-strand breaks (DSBs) and remains covalently linked to each 5’ end. The Mre11-Rad50-Nbs1 (MRN)-Ctp1 complex endonucleolytically removes Rec12 covalently linked to a short oligonucleotide (Rec12-oligo), an activity called “clipping.” The recessed 5’ end is then further digested, an activity called “resection,” to form a long 3’ single-stranded DNA tail that forms with intact DNA a joint DNA molecule to continue DSB repair. We concluded that most of the dozen ctp1 point mutants we studied retained nearly wild-type levels of resection but little clipping activity during meiosis. These conclusions were based on concordant genetic and physical analyses. Although we isolated these mutants based on a mitotic screen, we drew all of our conclusions from meiotic data.

In our physical assays for clipping, we found in wild-type cell extracts abundant Rec12-oligos, which were absent or barely detectable in ctp1 mutant extracts, either point mutant or complete deletion (ctp1Δ). Failure to clip off Rec12 leaves irreparable DSBs and consequently inviable spores; the ctp1 point mutants had 10- to 30,000-fold, and ctp1Δ 100,000-fold, reductions of viable spore yields compared to wild type. These results indicate that the ctp1 mutants have strongly reduced clipping activity.

To assay resection, we induced in meiotic cells a DSB at a well-defined site, using either the I-SceI or the I-PpoI homing endonuclease. Physical assays using Southern blots showed that resection of the DSB end proceeded as rapidly and extensively in the ctp1 point mutants as in wild type but slowly and to a much lesser extent in the ctp1Δ mutant. The homing endonucleases form DSBs without a covalently bound protein; I-SceI-dependent recombination thus requires resection but not clipping. In the ctp1 point mutants, the frequency of this recombination was equal to, or even twice as high as, that in ctp1+ cells, which was ~5 times higher than that in the ctp1Δ mutant. Thus, both physical and genetic assays indicate that resection is robust in the ctp1 point mutants.

In interpreting these results, it is important to consider the role of Exonuclease I (ExoI), which could potentially resect DSB ends and produce recombinants. The MRN complex blocks ExoI access to DSBs in meiotic cells, but the Ku complex blocks ExoI in mitotic cells. Thus, ExoI plays no apparent role in DSB repair in meiotic cells with an intact MRN complex; instead, MRN promotes access of Ctp1 to DSB ends. For example, in meiotic cells exoIΔ has no significant effect on I-SceI-dependent recombination, but ctp1Δ reduces it by a factor of ~5 (3). Conversely, in rad50Δ mutants ctp1Δ has no significant effect, but exoIΔ reduces recombination by a factor of ~4. As expected, the ctp1Δ exoIΔ double mutant is like ctp1Δ in rad50+ cells but like exoIΔ in rad50Δ mutants.

Jensen and Russell (2) assessed the mitotic activity of two of our ctp1 point mutants, along with ctp1+ and ctp1Δ, by analyzing the number and sizes of colonies formed on agar plates containing DNA damaging agents and spotted with 5-fold serial dilutions of cell cultures. Their results, like ours, showed that ctp1-6 and ctp1-25 are more DNA damage-resistant than the ctp1Δ mutant, indicating that the point mutants retain some activity. Removal of the Ku complex, by pku80Δ, suppressed these sensitivities but only if ExoI was present. Removal of ExoI enhanced the sensitivity to some agents (e.g., methyl methanesulfonate) but not, or only slightly, to others (e.g., ionizing radiation). In each of these various combinations, the point mutants were more resistant than the ctp1Δ mutant, confirming our results that the point mutants retain some activity. In nine out of ten cases, the ctp1-6 exoIΔ and ctp1-25 exoIΔ mutants were more resistant than ctp1Δ exoIΔ, which was completely sensitive to the agents tested. This result shows that the ctp1 point mutants retain an activity that can be supplied by ExoI, which we take to be resection.

Jensen and Russell proposed that the ctp1 point mutations reduce, but do not abolish, a single activity. They further proposed that this residual activity is sufficient to repair a single DSB per cell (as in the I-SceI and I-PpoI experiments we reported) but not multiple DSBs per cell [as in the experiments with wild-type Rec12, which makes about 60 DSBs per meiotic cell (4)]. This proposal is hard to reconcile with our physical assays of clipping and resection noted above. It is not clear to us how a single Ctp1 activity could be altered, in either KM or kcat, to resect one DSB in a cell with wild-type kinetics but not clip Rec12 off 60 DSBs in a cell if resection and clipping result from the same activity, since both processes take about the same amount of time. Rather, we think the mutant proteins have greater reductions of clipping activity than of resection activity. Reduction of the number of Ctp1 molecules per cell, from 60 or more to about 1, could account for our results, but we consider this an unlikely explanation for the many mutants we studied. Instead, we think our mutants have retained nearly wild-type levels of resection but have strongly reduced levels of clipping, in meiotic cells. Whether the active sites for these two activities are in Ctp1, the MRN complex, or both is not addressed by our experiments. Without tests of a very large number of Ctp1 mutants under many conditions, we think the conclusion that the two activities are not genetically separable is unwarranted.

Lijuan Ma, Neta Milman, Mridula Nambiar, and Gerald R. Smith

References:

1. Ma, L., Milman, N., Nambiar, M. and Smith, G.R. (2015) Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair. Nucleic Acids Res., 43, 7349-7359.

2. Jensen, K.L. and Russell, P. (2016) Ctp1-dependent clipping and resection of DNA double-strand breaks by Mre11 endonuclease complex are not genetically separable. Nucleic Acids Res., 44, 8241-8249.

3. Farah, J.A., Cromie, G.A. and Smith, G.R. (2009) Ctp1 and Exonuclease 1, alternative nucleases regulated by the MRN complex, are required for efficient meiotic DNA repair and recombination. Proc. Natl. Acad. Sci. USA, 106, 9356-9361.

4. Fowler, K.R., Sasaki, M., Milman, N., Keeney, S. and Smith, G.R. (2014) Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome. Genome Res., 24, 1650-1664.

On 2017 Jun 09, GERALD SMITH commented:

In Ma et al. (1), we isolated Schizosaccharomyces pombe ctp1 point mutations, which we interpreted to separate genetically two known meiotic activities that depend on Ctp1 for meiotic DNA break repair. A later paper by Jensen and Russell (2) claimed these activities are not genetically separable. We believe the disparity in interpretation stems largely from the use of meiotic cells in our studies but mitotic cells by Jensen and Russell. We maintain that in meiotic cells the activities are genetically separable.

During S. pombe meiosis, Rec12 (Spo11 homolog) makes DNA double-strand breaks (DSBs) and remains covalently linked to each 5’ end. The Mre11-Rad50-Nbs1 (MRN)-Ctp1 complex endonucleolytically removes Rec12 covalently linked to a short oligonucleotide (Rec12-oligo), an activity called “clipping.” The recessed 5’ end is then further digested, an activity called “resection,” to form a long 3’ single-stranded DNA tail that forms with intact DNA a joint DNA molecule to continue DSB repair. We concluded that most of the dozen ctp1 point mutants we studied retained nearly wild-type levels of resection but little clipping activity during meiosis. These conclusions were based on concordant genetic and physical analyses. Although we isolated these mutants based on a mitotic screen, we drew all of our conclusions from meiotic data.

In our physical assays for clipping, we found in wild-type cell extracts abundant Rec12-oligos, which were absent or barely detectable in ctp1 mutant extracts, either point mutant or complete deletion (ctp1Δ). Failure to clip off Rec12 leaves irreparable DSBs and consequently inviable spores; the ctp1 point mutants had 10- to 30,000-fold, and ctp1Δ 100,000-fold, reductions of viable spore yields compared to wild type. These results indicate that the ctp1 mutants have strongly reduced clipping activity.

To assay resection, we induced in meiotic cells a DSB at a well-defined site, using either the I-SceI or the I-PpoI homing endonuclease. Physical assays using Southern blots showed that resection of the DSB end proceeded as rapidly and extensively in the ctp1 point mutants as in wild type but slowly and to a much lesser extent in the ctp1Δ mutant. The homing endonucleases form DSBs without a covalently bound protein; I-SceI-dependent recombination thus requires resection but not clipping. In the ctp1 point mutants, the frequency of this recombination was equal to, or even twice as high as, that in ctp1+ cells, which was ~5 times higher than that in the ctp1Δ mutant. Thus, both physical and genetic assays indicate that resection is robust in the ctp1 point mutants.

In interpreting these results, it is important to consider the role of Exonuclease I (ExoI), which could potentially resect DSB ends and produce recombinants. The MRN complex blocks ExoI access to DSBs in meiotic cells, but the Ku complex blocks ExoI in mitotic cells. Thus, ExoI plays no apparent role in DSB repair in meiotic cells with an intact MRN complex; instead, MRN promotes access of Ctp1 to DSB ends. For example, in meiotic cells exoIΔ has no significant effect on I-SceI-dependent recombination, but ctp1Δ reduces it by a factor of ~5 (3). Conversely, in rad50Δ mutants ctp1Δ has no significant effect, but exoIΔ reduces recombination by a factor of ~4. As expected, the ctp1Δ exoIΔ double mutant is like ctp1Δ in rad50+ cells but like exoIΔ in rad50Δ mutants.

Jensen and Russell (2) assessed the mitotic activity of two of our ctp1 point mutants, along with ctp1+ and ctp1Δ, by analyzing the number and sizes of colonies formed on agar plates containing DNA damaging agents and spotted with 5-fold serial dilutions of cell cultures. Their results, like ours, showed that ctp1-6 and ctp1-25 are more DNA damage-resistant than the ctp1Δ mutant, indicating that the point mutants retain some activity. Removal of the Ku complex, by pku80Δ, suppressed these sensitivities but only if ExoI was present. Removal of ExoI enhanced the sensitivity to some agents (e.g., methyl methanesulfonate) but not, or only slightly, to others (e.g., ionizing radiation). In each of these various combinations, the point mutants were more resistant than the ctp1Δ mutant, confirming our results that the point mutants retain some activity. In nine out of ten cases, the ctp1-6 exoIΔ and ctp1-25 exoIΔ mutants were more resistant than ctp1Δ exoIΔ, which was completely sensitive to the agents tested. This result shows that the ctp1 point mutants retain an activity that can be supplied by ExoI, which we take to be resection.

Jensen and Russell proposed that the ctp1 point mutations reduce, but do not abolish, a single activity. They further proposed that this residual activity is sufficient to repair a single DSB per cell (as in the I-SceI and I-PpoI experiments we reported) but not multiple DSBs per cell [as in the experiments with wild-type Rec12, which makes about 60 DSBs per meiotic cell (4)]. This proposal is hard to reconcile with our physical assays of clipping and resection noted above. It is not clear to us how a single Ctp1 activity could be altered, in either KM or kcat, to resect one DSB in a cell with wild-type kinetics but not clip Rec12 off 60 DSBs in a cell if resection and clipping result from the same activity, since both processes take about the same amount of time. Rather, we think the mutant proteins have greater reductions of clipping activity than of resection activity. Reduction of the number of Ctp1 molecules per cell, from 60 or more to about 1, could account for our results, but we consider this an unlikely explanation for the many mutants we studied. Instead, we think our mutants have retained nearly wild-type levels of resection but have strongly reduced levels of clipping, in meiotic cells. Whether the active sites for these two activities are in Ctp1, the MRN complex, or both is not addressed by our experiments. Without tests of a very large number of Ctp1 mutants under many conditions, we think the conclusion that the two activities are not genetically separable is unwarranted.

Lijuan Ma, Neta Milman, Mridula Nambiar, and Gerald R. Smith

References:

1. Ma, L., Milman, N., Nambiar, M. and Smith, G.R. (2015) Two separable functions of Ctp1 in the early steps of meiotic DNA double-strand break repair. Nucleic Acids Res., 43, 7349-7359.

2. Jensen, K.L. and Russell, P. (2016) Ctp1-dependent clipping and resection of DNA double-strand breaks by Mre11 endonuclease complex are not genetically separable. Nucleic Acids Res., 44, 8241-8249.

3. Farah, J.A., Cromie, G.A. and Smith, G.R. (2009) Ctp1 and Exonuclease 1, alternative nucleases regulated by the MRN complex, are required for efficient meiotic DNA repair and recombination. Proc. Natl. Acad. Sci. USA, 106, 9356-9361.

4. Fowler, K.R., Sasaki, M., Milman, N., Keeney, S. and Smith, G.R. (2014) Evolutionarily diverse determinants of meiotic DNA break and recombination landscapes across the genome. Genome Res., 24, 1650-1664.