Mec1-independent activation of the Rad53 checkpoint kinase revealed by quantitative analysis of protein localization dynamics
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eLife Assessment:
In addition to identifying several components regulated by checkpoint kinases, the authors identify a novel non-canonical activation mode for the central checkpoint kinase Rad53, a phosphorylation event that does not depend on Mec1 and instead depends on proteins involved in retrograde signaling through Rtg3. The study thus reveals unanticipated complexities in the DNA replication stress response. Overall, the work is well done and the data support the main conclusions.
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Abstract
The replication checkpoint is essential for accurate DNA replication and repair, and maintenance of genomic integrity when a cell is challenged with genotoxic stress. Several studies have defined the complement of proteins that change subcellular location in the budding yeast Saccharomyces cerevisiae following chemically induced DNA replication stress using methyl methanesulfonate (MMS) or hydroxyurea (HU). How these protein movements are regulated remains largely unexplored. We find that the essential checkpoint kinases Mec1 and Rad53 are responsible for regulating the subcellular localization of 159 proteins during MMS-induced replication stress. Unexpectedly, Rad53 regulation of the localization of 52 proteins is independent of its known kinase activator Mec1, and in some scenarios independent of Tel1 or the mediator proteins Rad9 and Mrc1. We demonstrate that Rad53 is phosphorylated and active following MMS exposure in cells lacking Mec1 and Tel1. This noncanonical mode of Rad53 activation depends partly on the retrograde signaling transcription factor Rtg3, which also facilitates proper DNA replication dynamics. We conclude that there are biologically important modes of Rad53 protein kinase activation that respond to replication stress and operate in parallel to Mec1 and Tel1.
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Author Response
Reviewer #2 (Public Review):
Using an approach that combines synthetic genetic array (SGA) analysis with high-throughput microscopic analysis of the GFP-tagged yeast ORF collection in the budding yeast, Saccharomyces cerevisiae, this study has examined the contribution of the critical checkpoint kinases Mec1 and Rad53 to the subcellular relocalization of 322 candidate proteins in response to HU- and MMS-induced replication stress. Previous studies have established that Mec1 is required for Rad53 activation during replication stress and that Mec1 also serves checkpoint functions independent of Rad53. Unexpectedly, this study identifies groups of proteins whose stress-induced relocalization is dependent on Rad53 but not Mec1. This data indicates that Rad53 mediates some replication stress responses in a non-canonical …
Author Response
Reviewer #2 (Public Review):
Using an approach that combines synthetic genetic array (SGA) analysis with high-throughput microscopic analysis of the GFP-tagged yeast ORF collection in the budding yeast, Saccharomyces cerevisiae, this study has examined the contribution of the critical checkpoint kinases Mec1 and Rad53 to the subcellular relocalization of 322 candidate proteins in response to HU- and MMS-induced replication stress. Previous studies have established that Mec1 is required for Rad53 activation during replication stress and that Mec1 also serves checkpoint functions independent of Rad53. Unexpectedly, this study identifies groups of proteins whose stress-induced relocalization is dependent on Rad53 but not Mec1. This data indicates that Rad53 mediates some replication stress responses in a non-canonical manner that is independent of Mec1.
The authors confirm their initial observations from the screening approach by focusing on the Rad53-dependent and Mec1-independent focus formation of GFP-Rad54. Moreover, using mass-spec analysis the authors demonstrate that some Rad53 phosphorylation sites known to be critical for Rad53 activation, including a consensus Mec1 phosphorylation site, are phosphorylated after replication stress even in the absence of Mec1. Motivated by this finding the authors screen for potential kinase and phosphatase pathways that may regulate Rad53 function during MMS-induced replication stress. Top hits identified include members of the retrograde signaling pathway, which is confirmed by conventional genetic assays while mass spec analysis supports the involvement of Rtg3 in mediating Rad53 phosphorylation during replication stress in the absence of Mec1.
Overall this is a solid study reporting unexpected new findings that significantly advance our view of the global replication checkpoint response. The data are generally of high quality, well presented and quantified, and overall support the authors' claims. The mass spec approach used here to identify Rad53 phosphorylation sites offers an unbiased alternative to the simpler and more widely employed gel-shift method to monitor Rad53 activation. The hits identified in the various screens presented here provide a platform for potential follow-up studies by the community. The main drawback is that it remains unclear how Rtg3 promotes Rad53 activation. However, this could be considered to be beyond the scope of this study.
We thank the reviewer for their positive assessment of our experimental data. We have made the changes requested by the reviewer to increase the clarity of Figure 5, and performed a second replicate to show that the FACS data are reproducible.
Reviewer #3 (Public Review):
The work by Ho et al describes the identification of Mec1/Tel1 independent activation of Rad53 after MMS treatment, which could lead to changes of GFP fusion signals for several dozens of proteins and this was partly dependent on Rtg3. Starting from an unbiased, targeted screen, the authors identified proteins whose GFP fusion signals changed intensity in rad53∆ but not in mec1∆ cells using live cell imaging, including Rad54. Using Rad54 as a readout for the subsequent experiments, a second screen amongst kinases/phosphatases and their regulators found that rtg2-3 mutants reduced Rad54-GFP intensity. Mass spectrometry data identified Rad53 phosphorylate sites in mec1∆ tel1∆ cells, consistent with the cell biological data described above. Overall, the work was well done and supported the main conclusions. The concept of Mec1/Tel1-independent and Rtg3-dependent Rad53 activation connects checkpoint signaling with the retrograde pathway.
We thank the reviewer for their positive assessment of our experimental work and appreciate the suggestions that ultimately led to interesting new data. As outlined below, we attempted to perform most of the experiments suggested by the reviewer. Unfortunately, experiments with a MEC1-AID degron allele were inconclusive, with details summarized below. However, we have identified additional proteins whose re-localization is affected by Rtg3, providing stronger support for its role in the replication stress response. We revised the manuscript to add these new details.
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eLife Assessment:
In addition to identifying several components regulated by checkpoint kinases, the authors identify a novel non-canonical activation mode for the central checkpoint kinase Rad53, a phosphorylation event that does not depend on Mec1 and instead depends on proteins involved in retrograde signaling through Rtg3. The study thus reveals unanticipated complexities in the DNA replication stress response. Overall, the work is well done and the data support the main conclusions.
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Reviewer #1 (Public Review):
The DNA damage checkpoint is a cellular signalling pathway that responds to DNA damage and replication stress. This manuscript by Ho et al. systematically investigates an aspect of the checkpoint response in budding yeast that has been previously understudied, namely which proteins change subcellular and how these changed depend on the checkpoint kinases Mec1 and Rad53. By nice detective work the authors find a new mode of activation of Rad53, which is Mec1-independent, but rather depends on factors of so called retrograde signalling. Currently, we view checkpoint signalling as hierarchical, with Mec1 and Tel1 activating Rad53, despite both Mec1 and Rad53 having independent targets. This manuscript challenges that view by finding a Mec1 (and Tel1) independent mode of activation. It is very clear from …
Reviewer #1 (Public Review):
The DNA damage checkpoint is a cellular signalling pathway that responds to DNA damage and replication stress. This manuscript by Ho et al. systematically investigates an aspect of the checkpoint response in budding yeast that has been previously understudied, namely which proteins change subcellular and how these changed depend on the checkpoint kinases Mec1 and Rad53. By nice detective work the authors find a new mode of activation of Rad53, which is Mec1-independent, but rather depends on factors of so called retrograde signalling. Currently, we view checkpoint signalling as hierarchical, with Mec1 and Tel1 activating Rad53, despite both Mec1 and Rad53 having independent targets. This manuscript challenges that view by finding a Mec1 (and Tel1) independent mode of activation. It is very clear from survival and mass spectrometry data that in the absence of Mec1 this activation pathway and Rtg3 has a key role in activating Rad53. In the current form of the manuscript, it remains however difficult to assess what is the contribution of these factors on Rad53 activation in an otherwise WT background.
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Reviewer #2 (Public Review):
Using an approach that combines synthetic genetic array (SGA) analysis with high-throughput microscopic analysis of the GFP-tagged yeast ORF collection in the budding yeast, Saccharomyces cerevisiae, this study has examined the contribution of the critical checkpoint kinases Mec1 and Rad53 to the subcellular relocalization of 322 candidate proteins in response to HU- and MMS-induced replication stress. Previous studies have established that Mec1 is required for Rad53 activation during replication stress and that Mec1 also serves checkpoint functions independent of Rad53. Unexpectedly, this study identifies groups of proteins whose stress-induced relocalization is dependent on Rad53 but not Mec1. This data indicates that Rad53 mediates some replication stress responses in a non-canonical manner that is …
Reviewer #2 (Public Review):
Using an approach that combines synthetic genetic array (SGA) analysis with high-throughput microscopic analysis of the GFP-tagged yeast ORF collection in the budding yeast, Saccharomyces cerevisiae, this study has examined the contribution of the critical checkpoint kinases Mec1 and Rad53 to the subcellular relocalization of 322 candidate proteins in response to HU- and MMS-induced replication stress. Previous studies have established that Mec1 is required for Rad53 activation during replication stress and that Mec1 also serves checkpoint functions independent of Rad53. Unexpectedly, this study identifies groups of proteins whose stress-induced relocalization is dependent on Rad53 but not Mec1. This data indicates that Rad53 mediates some replication stress responses in a non-canonical manner that is independent of Mec1.
The authors confirm their initial observations from the screening approach by focusing on the Rad53-dependent and Mec1-independent focus formation of GFP-Rad54. Moreover, using mass-spec analysis the authors demonstrate that some Rad53 phosphorylation sites known to be critical for Rad53 activation, including a consensus Mec1 phosphorylation site, are phosphorylated after replication stress even in the absence of Mec1. Motivated by this finding the authors screen for potential kinase and phosphatase pathways that may regulate Rad53 function during MMS-induced replication stress. Top hits identified include members of the retrograde signaling pathway, which is confirmed by conventional genetic assays while mass spec analysis supports the involvement of Rtg3 in mediating Rad53 phosphorylation during replication stress in the absence of Mec1.
Overall this is a solid study reporting unexpected new findings that significantly advance our view of the global replication checkpoint response. The data are generally of high quality, well presented and quantified, and overall support the authors' claims. The mass spec approach used here to identify Rad53 phosphorylation sites offers an unbiased alternative to the simpler and more widely employed gel-shift method to monitor Rad53 activation. The hits identified in the various screens presented here provide a platform for potential follow-up studies by the community. The main drawback is that it remains unclear how Rtg3 promotes Rad53 activtation. However, this could be considered to be beyond the scope of this study.
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Reviewer #3 (Public Review):
The work by Ho et al describes the identification of Mec1/Tel1 independent activation of Rad53 after MMS treatment, which could lead to changes of GFP fusion signals for several dozens of proteins and this was partly dependent on Rtg3. Starting from an unbiased, targeted screen, the authors identified proteins whose GFP fusion signals changed intensity in rad53∆ but not in mec1∆ cells using live cell imaging, including Rad54. Using Rad54 as a readout for the subsequent experiments, a second screen amongst kinases/phosphatases and their regulators found that rtg2-3 mutants reduced Rad54-GFP intensity. Mass spectrometry data identified Rad53 phosphorylate sites in mec1∆ tel1∆ cells, consistent with the cell biological data described above. Overall, the work was well done and supported the main conclusions. The …
Reviewer #3 (Public Review):
The work by Ho et al describes the identification of Mec1/Tel1 independent activation of Rad53 after MMS treatment, which could lead to changes of GFP fusion signals for several dozens of proteins and this was partly dependent on Rtg3. Starting from an unbiased, targeted screen, the authors identified proteins whose GFP fusion signals changed intensity in rad53∆ but not in mec1∆ cells using live cell imaging, including Rad54. Using Rad54 as a readout for the subsequent experiments, a second screen amongst kinases/phosphatases and their regulators found that rtg2-3 mutants reduced Rad54-GFP intensity. Mass spectrometry data identified Rad53 phosphorylate sites in mec1∆ tel1∆ cells, consistent with the cell biological data described above. Overall, the work was well done and supported the main conclusions. The concept of Mec1/Tel1-independent and Rtg3-dependent Rad53 activation connects checkpoint signaling with the retrograde pathway.
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