2022 Fiscal Year Annual Research Report
Elucidation of mechanisms ensuring optimal double-strand break number in meiotic cells
| Project/Area Number |
21H02400
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| Research Institution | Kyoto University |
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Principal Investigator |
Carlton Peter 京都大学, 生命科学研究科, 准教授 (20571813)
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | 減数分裂 / 線虫 / 染色体 / 二重鎖切断 / キナーゼ / ホスファターゼ |
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| Outline of Annual Research Achievements |
We have continued to address the mechanisms of meiotic double-strand break (DSB) control by phosphoregulation of the protein DSB-1 in this fiscal year.
We found that mutating a single serine residue in DSB-1, S186, to alanine, leads to hyperactive DSB formation, even in the absence of the protein DSB-2. Loss of DSB-2 norally leads to high embryonic inviability due to loss of DSBs. Since loss of DSB-2 can be rescued so easily, we reasoned that it might be required for long-term genome stability. We therefore have performed whole-genome long-read sequencing on DSB-1 non-phosphorylatable mutant lines that have been experiencing high DSB levels for over 50 generations. While analysis of this sequence information is still in progress, individual mutant animals have been isolated with spontaneous mutations and spontaneous tetraploidization. Since spontaneous tetraploidization is never observed in wild-type animals, it is plausible that the genome has become less stable in the non-phosphorylatable DSB-1 mutant animals.
Our attempts to directly demonstrate phosphorylation of DSB-1 by phosphospecific antibody production or mass spectrometry have not yet been successful; however, we have now completed construction of a FLAG-tagged DSB-1 allele with full viability that is very clean on a western blot; this reagent should greatly aid in our further efforts to characterize DSB-1.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
A major output from this proposal was published in FY2022, in the journal Elife. In the published version, we included two additional figures: one showing the AlphaFold2 prediction of a trimerization interface between the proteins DSB-1, DSB-2, and DSB-3; and one showing a phylogenetic analysis of DSB-1 and DSB-2 that demonstrates DSB-1 is closer to the ancestral version of the protein (known as Rec114 in most species), while DSB-2 has diverged in sequence, and thus perhaps in function as well.
The prediction has since been confirmed in other species (yeast and mouse). Work since this manuscript's submission focuses on interactions between DSB-1 and the SPO-11 core complex, long-term genome stability, and the actual mechanism leading from DSB-1 phosphorylation to reduction of DSBs, and it is likely to lead to another submitted manuscript within or soon after this project's end.
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| Strategy for Future Research Activity |
We are testing directly whether the C-terminal extension of SPO-11 specific to the Elegans group of Caenorhabditis nematodes replaces the protein TOPVIBL as the interactor of DSB-1, which will demonstrate the flexibility of the DSB control system in eukaryotes as well as shed further light on mechanisms of phosphoregulatory control of cutting by SPO-11. To this end, we have constructed tagged C-terminal deletion strains that show normal protein expression and loss of DSBs, in agreement with the hypothesis. We have also noted that the critical S186 residue in DSB-1 lies in a conserved recognition motif (FXXP) for the PP4 phosphatase holoenzyme, raising the possibility that phosphorylation there is blocking the ability of PP4 to dephosphorylate further sites on DSB-1. We will construct mutations that leave S186 intact but disrupt the predicted binding of PP4, and test whether DSB-1 becomes further phosphorylated in this condition.
Further, building on a findings in recent publication that loss of DSB control in mouse leads to genome rearrangement at meiotic DSB hotspots in only a few generations, we are analyzing long-read sequence data of animals with non-phosphorylatable mutations in DSB-1 to assess the presence of rearrangements, deletions, and other evidence of genome instability.
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