Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity
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Evaluation Summary:
The connection between double-strand break (DSB) formation and chromosome pairing/synapsis during meiosis is not fully understood. In this paper, the authors show that the formation of DSBs is regulated by the DNA damage response (DDR) machinery. The paper will be of interest to the broad meiosis and DDR communities. While the main conclusions of the manuscript appear to be well-supported by the data, some gaps are present and the manuscript would therefore benefit from additional (mostly minor) changes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated to be neither too high nor too low. Here, we show that in Caenorhabditis elegans , meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4 PPH-4.1 phosphatase and ATR ATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants and in the wild-type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4 PPH-4.1 , ATR ATL-1 , and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.
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Author Response
Reviewer #2 (Public Review):
In this paper, Guo et al. investigated how DNA double-strand break (DSB) formation is regulated during C. elegans meiosis. Meiotic recombination initiates with programmed DSB formation, which is catalyzed by the Spo11 holoenzyme. In C. elegans, three SPO-11 cofactors have been identified so far. One of them is DSB-1, which is one of two homologs of Rec114. The authors first show that a phosphorylated form of DSB-1 appears as a slower migrating species on western blots. Using this as a readout, they demonstrate that phosphorylation of DSB-1 is dependent on two DNA damage sensor kinases, ATR (ATL-1) and ATM (ATM-1) and that dephosphorylation of DSB-1 is partially mediated by a member of PP4 phosphatase, PPH-4.1. It was previously shown that PPH-4.1 is required for multiple steps in meiotic …
Author Response
Reviewer #2 (Public Review):
In this paper, Guo et al. investigated how DNA double-strand break (DSB) formation is regulated during C. elegans meiosis. Meiotic recombination initiates with programmed DSB formation, which is catalyzed by the Spo11 holoenzyme. In C. elegans, three SPO-11 cofactors have been identified so far. One of them is DSB-1, which is one of two homologs of Rec114. The authors first show that a phosphorylated form of DSB-1 appears as a slower migrating species on western blots. Using this as a readout, they demonstrate that phosphorylation of DSB-1 is dependent on two DNA damage sensor kinases, ATR (ATL-1) and ATM (ATM-1) and that dephosphorylation of DSB-1 is partially mediated by a member of PP4 phosphatase, PPH-4.1. It was previously shown that PPH-4.1 is required for multiple steps in meiotic chromosome dynamics, such as homolog pairing, synapsis, and DSB formation. Interestingly, heterozygous null mutation of atl-1, but not atm-1 deletion, restores meiotic DSB formation in pph-4.1 animals. It was further shown that DSB-1 contains five S/T-Q sites within its disordered region, and mutating all five sites leads to increased DSB formation and partially restores homologous pairing, DSB formation, and chiasma formation in pph-4.1 mutants. The rescue of homolog pairing was unexpected, and this illustrates a requirement of meiotic DSBs in enforcing correct pairing in C. elegans, similar to the case in other eukaryotes. The authors further demonstrate that DSB-1 phosphorylation occurs in an age-dependent manner, and this trend is not observed in dsb-2 mutants, which leads to a proposal that DSB-2 might have evolved to compensate for the decreased activity of the phosphorylated DSB-1 in older animals.
Overall, this is a nice study illustrating the antagonistic relationship between ATL-1and PPH-4.1 in regulating meiotic DSB formation. This work establishes that meiotic DSB formation is negatively regulated by ATL-1 in C. elegans, similar to what has been established in other organisms, and adds that a PP4 family member opposes this function of ATR. Rescued DSB formation and homolog pairing in pph-4.1; dsb-1(5A) is striking (even though it's partial), indicating that DSB-1 is a major target of PPH-4.1. Perhaps the partial rescue is somewhat expected, as PPH-4.1 is involved in many meiotic processes other than DSB formation. Therefore, more thorough analyses of the "rescued" phenotypes in pph-4.1 mutants, especially the status of SC assembly in both pph-4.1; dsb-1(phosphomutant series) and irradiated animals (with different doses), will help clarify some of the discussion points regarding the function of PPH-4.1 in processing recombination intermediates and the degree to which exogenous DSBs contribute to homolog pairing and synapsis in C. elegans. Another criticism is that this study only focuses on the phosphoregulation of DSB-1, while both DSB-1 and DSB-2 are C. elegans homologs of Rec114, and DSB-2 also contains four S/T-Q sites. Structural prediction of the putative DSB-1:DSB-2:DSB-3 and DSB-1:DSB-1:DSB-3 complexes in the discussion is very illuminating and suggests that perhaps the remaining DSB-1 in dsb-2 mutants is the pool that forms the DSB-1:DSB-1:DSB-3 complex and is prematurely phosphorylated by ATL-1 simply because of mass action. A model figure illustrating the phospho-regulation of DSB-1 (and maybe DSB-2) by ATL-1 and PPH-4.1 will greatly strengthen this paper.
We have now examined the status of SC assembly in pph-4.1 combined with dsb-1 or atl-1/nT1 mutations and included in the data as discussed in response to the specific question #4 as below. Further, to expand our understanding of possible phosphoregulation on DSB-2, we have generated and examined dsb-2 non-phosphorylatable mutants (4A) at its SQ sites. In contrast to dsb-1 non-phosphorylatable mutations, the dsb-2 (4A) mutation did not increase the number of DSBs, suggesting that DSB-2 is refractory to phosphoregulation. We now include this data in Figure 2–figure supplement 2. We also include a model figure of DSB-1 phosphoregulation in Figure 6B as suggested.
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Evaluation Summary:
The connection between double-strand break (DSB) formation and chromosome pairing/synapsis during meiosis is not fully understood. In this paper, the authors show that the formation of DSBs is regulated by the DNA damage response (DDR) machinery. The paper will be of interest to the broad meiosis and DDR communities. While the main conclusions of the manuscript appear to be well-supported by the data, some gaps are present and the manuscript would therefore benefit from additional (mostly minor) changes.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, Guo et al. seek to understand how DSB formation is regulated during C. elegans meiosis. Building upon previous findings (including the lab's own previous published results), the authors focused on the balance between kinase and phosphatase activity on DSB-1, a key DSB protein and orthologue of the SPO-11 co-factor Rec114
Using a combination of genetic approaches, imaging and phospho-induced electrophoretic mobility shifts, the authors show that phosphorylation of DSB-1 is mediated by two major kinases, ATR and ATM, and is counteracted by the PP4 phosphatase, PPH-4.1. While PPH-4.1 was known to be involved in pairing, synapsis and generation of DSB, the authors report that an atl-1 (ATR) null (partially) rescues the DSB defect in pph-4.1 mutants. In line with this, a DSB-1 phospho-mutant …
Reviewer #1 (Public Review):
In this manuscript, Guo et al. seek to understand how DSB formation is regulated during C. elegans meiosis. Building upon previous findings (including the lab's own previous published results), the authors focused on the balance between kinase and phosphatase activity on DSB-1, a key DSB protein and orthologue of the SPO-11 co-factor Rec114
Using a combination of genetic approaches, imaging and phospho-induced electrophoretic mobility shifts, the authors show that phosphorylation of DSB-1 is mediated by two major kinases, ATR and ATM, and is counteracted by the PP4 phosphatase, PPH-4.1. While PPH-4.1 was known to be involved in pairing, synapsis and generation of DSB, the authors report that an atl-1 (ATR) null (partially) rescues the DSB defect in pph-4.1 mutants. In line with this, a DSB-1 phospho-mutant lacking five S/T-Q sites rescues the homologous pairing and synapsis defect of pph-4.1 mutants. These results argue in favour of the notion that DSBs strengthen synapsis in C. elegans.
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Reviewer #2 (Public Review):
In this paper, Guo et al. investigated how DNA double-strand break (DSB) formation is regulated during C. elegans meiosis. Meiotic recombination initiates with programmed DSB formation, which is catalyzed by the Spo11 holoenzyme. In C. elegans, three SPO-11 cofactors have been identified so far. One of them is DSB-1, which is one of two homologs of Rec114. The authors first show that a phosphorylated form of DSB-1 appears as a slower migrating species on western blots. Using this as a readout, they demonstrate that phosphorylation of DSB-1 is dependent on two DNA damage sensor kinases, ATR (ATL-1) and ATM (ATM-1) and that dephosphorylation of DSB-1 is partially mediated by a member of PP4 phosphatase, PPH-4.1. It was previously shown that PPH-4.1 is required for multiple steps in meiotic chromosome dynamics, …
Reviewer #2 (Public Review):
In this paper, Guo et al. investigated how DNA double-strand break (DSB) formation is regulated during C. elegans meiosis. Meiotic recombination initiates with programmed DSB formation, which is catalyzed by the Spo11 holoenzyme. In C. elegans, three SPO-11 cofactors have been identified so far. One of them is DSB-1, which is one of two homologs of Rec114. The authors first show that a phosphorylated form of DSB-1 appears as a slower migrating species on western blots. Using this as a readout, they demonstrate that phosphorylation of DSB-1 is dependent on two DNA damage sensor kinases, ATR (ATL-1) and ATM (ATM-1) and that dephosphorylation of DSB-1 is partially mediated by a member of PP4 phosphatase, PPH-4.1. It was previously shown that PPH-4.1 is required for multiple steps in meiotic chromosome dynamics, such as homolog pairing, synapsis, and DSB formation. Interestingly, heterozygous null mutation of atl-1, but not atm-1 deletion, restores meiotic DSB formation in pph-4.1 animals. It was further shown that DSB-1 contains five S/T-Q sites within its disordered region, and mutating all five sites leads to increased DSB formation and partially restores homologous pairing, DSB formation, and chiasma formation in pph-4.1 mutants. The rescue of homolog pairing was unexpected, and this illustrates a requirement of meiotic DSBs in enforcing correct pairing in C. elegans, similar to the case in other eukaryotes. The authors further demonstrate that DSB-1 phosphorylation occurs in an age-dependent manner, and this trend is not observed in dsb-2 mutants, which leads to a proposal that DSB-2 might have evolved to compensate for the decreased activity of the phosphorylated DSB-1 in older animals.
Overall, this is a nice study illustrating the antagonistic relationship between ATL-1and PPH-4.1 in regulating meiotic DSB formation. This work establishes that meiotic DSB formation is negatively regulated by ATL-1 in C. elegans, similar to what has been established in other organisms, and adds that a PP4 family member opposes this function of ATR. Rescued DSB formation and homolog pairing in pph-4.1; dsb-1(5A) is striking (even though it's partial), indicating that DSB-1 is a major target of PPH-4.1. Perhaps the partial rescue is somewhat expected, as PPH-4.1 is involved in many meiotic processes other than DSB formation. Therefore, more thorough analyses of the "rescued" phenotypes in pph-4.1 mutants, especially the status of SC assembly in both pph-4.1; dsb-1(phosphomutant series) and irradiated animals (with different doses), will help clarify some of the discussion points regarding the function of PPH-4.1 in processing recombination intermediates and the degree to which exogenous DSBs contribute to homolog pairing and synapsis in C. elegans. Another criticism is that this study only focuses on the phosphoregulation of DSB-1, while both DSB-1 and DSB-2 are C. elegans homologs of Rec114, and DSB-2 also contains four S/T-Q sites. Structural prediction of the putative DSB-1:DSB-2:DSB-3 and DSB-1:DSB-1:DSB-3 complexes in the discussion is very illuminating and suggests that perhaps the remaining DSB-1 in dsb-2 mutants is the pool that forms the DSB-1:DSB-1:DSB-3 complex and is prematurely phosphorylated by ATL-1 simply because of mass action. A model figure illustrating the phospho-regulation of DSB-1 (and maybe DSB-2) by ATL-1 and PPH-4.1 will greatly strengthen this paper.
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Reviewer #3 (Public Review):
In this paper, Guo et al describe a novel function that modulates the frequency of meiotic double-strand breaks that initiate recombination through the inhibition by phosphorylation of one protein necessary for DSB formation, DSB-1.
Studies in several species (mammals, budding yeast, Drosophila) have described mechanisms that specifically limit the number of meiotic DSBs.This study performed in C. elegans reports convincingly that there is a balance between phosphorylation of DSB-1 (mainly by the ATL-1 (ATR) kinase) and its dephosphorylation by the PPH-4.1 (PP4) conserved phosphatase. Furthermore, they propose a mechanism by which DSB-1 phosphorylation is evolving with age, and how it is balanced by the activity of a related DSB protein, DSB-2. Finally, using structure prediction, they propose a mechanism …
Reviewer #3 (Public Review):
In this paper, Guo et al describe a novel function that modulates the frequency of meiotic double-strand breaks that initiate recombination through the inhibition by phosphorylation of one protein necessary for DSB formation, DSB-1.
Studies in several species (mammals, budding yeast, Drosophila) have described mechanisms that specifically limit the number of meiotic DSBs.This study performed in C. elegans reports convincingly that there is a balance between phosphorylation of DSB-1 (mainly by the ATL-1 (ATR) kinase) and its dephosphorylation by the PPH-4.1 (PP4) conserved phosphatase. Furthermore, they propose a mechanism by which DSB-1 phosphorylation is evolving with age, and how it is balanced by the activity of a related DSB protein, DSB-2. Finally, using structure prediction, they propose a mechanism for the relative activities of the related DSB-1 and DSB-2 proteins within the protein complex required for DSB formation.
All the involved proteins are conserved, and interestingly, the mechanisms described here are reminiscent of results obtained in budding yeast, where ATM/ATR phosphorylation targets the Rec114 homolog, which highlights the high degree of conservation of this important regulation.
Globally, the study is well performed, the conclusions are well supported by the experimental data, and both the conservation, but also the specific effect seen during aging, add value to this study and make it of broad interest, in particular to the meiosis and C. elegans communities.
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