Novel mechanistic insights into the role of Mer2 as the keystone of meiotic DNA break formation
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Evaluation Summary:
Using a combination of biochemical approaches and yeast genetics, the authors study the function of the DNA double-strand break factor Mer2. The authors show that Mer2 interacts with a meiotic chromosome axis factor (Hop1), nucleosomes, the nucleosome-binding protein Spp1, and the double-strand break factor Mre11 to serve as a "keystone" for meiotic DNA break formation. These findings represent an important step forward in understanding the functions of this highly conserved protein in meiosis.
(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 meiosis, DNA double-strand break (DSB) formation by Spo11 initiates recombination and enables chromosome segregation. Numerous factors are required for Spo11 activity, and couple the DSB machinery to the development of a meiosis-specific ‘axis-tethered loop’ chromosome organisation. Through in vitro reconstitution and budding yeast genetics, we here provide architectural insight into the DSB machinery by focussing on a foundational DSB factor, Mer2. We characterise the interaction of Mer2 with the histone reader Spp1, and show that Mer2 directly associates with nucleosomes, likely highlighting a contribution of Mer2 to tethering DSB factors to chromatin. We reveal the biochemical basis of Mer2 association with Hop1, a HORMA domain-containing chromosomal axis factor. Finally, we identify a conserved region within Mer2 crucial for DSB activity, and show that this region of Mer2 interacts with the DSB factor Mre11. In combination with previous work, we establish Mer2 as a keystone of the DSB machinery by bridging key protein complexes involved in the initiation of meiotic recombination.
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Evaluation Summary:
Using a combination of biochemical approaches and yeast genetics, the authors study the function of the DNA double-strand break factor Mer2. The authors show that Mer2 interacts with a meiotic chromosome axis factor (Hop1), nucleosomes, the nucleosome-binding protein Spp1, and the double-strand break factor Mre11 to serve as a "keystone" for meiotic DNA break formation. These findings represent an important step forward in understanding the functions of this highly conserved protein in meiosis.
(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):
The central process of meiotic cell division is the repair of induced double-strand breaks by meiotic recombination, which mediates the pairing of homologous chromosomes and their exchange of genetic material by crossing-over. In this study, Rousova et al aimed to determine the molecular basis of the essential function of Mer2 in regulating the double-strand break response in yeast meiosis. In this, they studied Mer2's interactions with Spp1, Hop1 and Mre11, in order to determine how Mer2 brings together nucleosomal DNA, the meiotic chromosome axis and the double-strand break resection machinery. Through a robust biochemical approach, they determined that Mer2 has a core tetrameric structure that recruits two Spp1 molecules in a high-affinity 4:2 complex. This complex interacts with nucleosomes in a manner …
Reviewer #1 (Public Review):
The central process of meiotic cell division is the repair of induced double-strand breaks by meiotic recombination, which mediates the pairing of homologous chromosomes and their exchange of genetic material by crossing-over. In this study, Rousova et al aimed to determine the molecular basis of the essential function of Mer2 in regulating the double-strand break response in yeast meiosis. In this, they studied Mer2's interactions with Spp1, Hop1 and Mre11, in order to determine how Mer2 brings together nucleosomal DNA, the meiotic chromosome axis and the double-strand break resection machinery. Through a robust biochemical approach, they determined that Mer2 has a core tetrameric structure that recruits two Spp1 molecules in a high-affinity 4:2 complex. This complex interacts with nucleosomes in a manner that depends on the induced dimerisation of Spp1 and is strengthened by an unanticipated direct interaction between Mer2 and nucleosomes. They further show that Mer2's core/C-terminal end binds directly to Hop1 in a manner that is inhibited by the closure interaction of Hop1's HORMA domain. Finally, they identify point mutations of conserved amino-acids within Mer2's N-terminus that block sporulation and impair double-strand break formation in vivo, thereby indicating their importance in this aspect of its function. These mutations weakened the Mer2-Mre11 interaction by yeast two-hybrid, and to a lesser extent through pull-down of recombinant proteins, suggesting that a weakening of the interaction between Mer2's N-terminus may at least in part explain the in vivo phenotype. Together, this paper provides important molecular insight into Mer2's interactions with three distinct components of double-strand break sites. These findings will provide essential foundations for ultimately uncovering the full three-dimensional structure of Mer2's ternary assembly at double-strand breaks and the precise molecular mechanism whereby it regulates the double-strand break machinery.
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Reviewer #2 (Public Review):
The study of eukaryotic meiosis has for many years been hampered by the inability to purify and reconstitute important meiotic DNA-binding and DNA cleavage/recombination factors, limiting the field's ability to perform detailed interaction analysis and structure-function dissection of the large set of proteins known to be important for meiotic recombination. Recent years have seen major advances in biochemical reconstitution of meiotic chromosome-associated proteins, and this manuscript is a prime example of what can be learned through this kind of analysis. The authors demonstrate direct interaction between Mer2 and: (1) Spp1 (a previously known interaction); (2) nucleosomes; (3) the axis protein Hop1; and (4) Mre11, a member of the conserved MRX/MRN complex required for DNA break formation. Despite limited …
Reviewer #2 (Public Review):
The study of eukaryotic meiosis has for many years been hampered by the inability to purify and reconstitute important meiotic DNA-binding and DNA cleavage/recombination factors, limiting the field's ability to perform detailed interaction analysis and structure-function dissection of the large set of proteins known to be important for meiotic recombination. Recent years have seen major advances in biochemical reconstitution of meiotic chromosome-associated proteins, and this manuscript is a prime example of what can be learned through this kind of analysis. The authors demonstrate direct interaction between Mer2 and: (1) Spp1 (a previously known interaction); (2) nucleosomes; (3) the axis protein Hop1; and (4) Mre11, a member of the conserved MRX/MRN complex required for DNA break formation. Despite limited purity for many of their Mer2 constructs, the authors nicely dissect these interactions and map them to different parts of the Mer2. A clear understanding of Mer2 clearly will require some structural analysis, especially of its tetrameric coiled-coil core region and its interactions with nucleosomes and other factors. But in the absence of 3D structure (which will be very difficult), the current study takes us a long way in understanding the roles of Mer2 in meiotic recombination.
While most of the conclusions are well-supported by the data, a few observations would benefit from either stronger in vitro data or supporting genetic data. In particular, the conclusion that Mer2 specifically interacts with "open/unlocked" Hop1 and not its closed form is very interesting, but the pulldown gel in Figure 4C is not 100% convincing due to the presence of faint bands in pulldown lanes (perhaps contaminants?). More importantly, the finding that Mer2 interact directly with Mre11 is exciting, but needs some additional support - potentially in the form of chromosome spreads showing Mre11 or its partners are not recruited in the identified 3A/4A mutants of Mer2.
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Reviewer #3 (Public Review):
The manuscript addresses the mechanistic roles of Mer2 in meiotic DNA double-stranded break (DSB) formation in budding yeast. Mer2 is a central component of the meiotic DNA break machinery. Accordingly, Mer2 has numerous known interactions that are thought to (i) promote assembly of the DSB machinery, (ii) promote anchoring of the DSB machinery to chromosome cores and (iii) enable the recruitment of DSB sites to the DSB machinery. The manuscript makes an impressive effort to comprehensively characterize protein interactions of Mer2 that are relevant for the listed functions in DNA break formation. The experiments are well executed and informative. The results are mainly derived from in vitro experiments which uncover potentially important new functions and mechanisms for Mer2. In particular, the newly …
Reviewer #3 (Public Review):
The manuscript addresses the mechanistic roles of Mer2 in meiotic DNA double-stranded break (DSB) formation in budding yeast. Mer2 is a central component of the meiotic DNA break machinery. Accordingly, Mer2 has numerous known interactions that are thought to (i) promote assembly of the DSB machinery, (ii) promote anchoring of the DSB machinery to chromosome cores and (iii) enable the recruitment of DSB sites to the DSB machinery. The manuscript makes an impressive effort to comprehensively characterize protein interactions of Mer2 that are relevant for the listed functions in DNA break formation. The experiments are well executed and informative. The results are mainly derived from in vitro experiments which uncover potentially important new functions and mechanisms for Mer2. In particular, the newly characterized interaction of Mer2 with nucleosomes and Hop1 will shape our models of meiotic recombination initiation. These discoveries will likely guide and necessitate future functional analysis.
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