| 研究実績の概要 |
We have shed light on the interaction between the double-strand break promoting factor DSB-1 and its interacting phosphoregulators: the DNA damage kinase ATR, and PPH-4.1 phosphatase. We showed that a GFP fusion to DSB-1 displays slow-migrating isoforms on a western blot, and the slower isoforms increase proportionally after either gamma-irradiation or depletion of PPH-4.1 protein, suggesting the slower-migrating forms are phosphorylated. After alkaline phosphatase treatment, the higher (slower) bands disappeared, indicating that they indeed phospho-forms of DSB-1. DSB-1 possesses five ATM/ATR consensus motifs (SQ) in its intrinsically disordered domain. We mutated all five of these to non-phosphorylatable Alanine, and found that the resulting DSB-1(5A) protein lost its band shift. Further, the 5A allele showed higher counts of RAD-51 foci in mid-meiotic prophase. In a pph-4.1 deletion background, the high number of foci induced by the dsb-1(5A) allele persists; in other words, DSB-1(5A) can rescue the double-strand break defect seen in pph-4.1 single mutants. Accordingly, the viability of pph-4.1; dsb-1(5A) double mutants was rescued to 41%, significantly higher than the ~2% viability of pph-4.1 single mutants. Further, we found that auxin-induced depletion of ATR kinase, but not ATM kinase, also led to increased double-strand breaks in pph-4.1 mutants, showing that ATR is the kinase that phosphorylates DSB-1 protein. Taken together, our results define DSB-1 as central to a critical pathway that implements negative feedback of meiotic double-strand breaks.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
We have completed our first manuscript which is currently available on bioRxiv and has undergone one round of peer review at the journal eLife. We are making revisions to the manuscript and expect publication by the end of July.
The droplet-like appearance of GFP-DSB-1 we described in our proposal was found to depend solely on the presence of GFP fusion protein; in other words, the droplet was likely artifactually induced by GFP. While we still think it likely that DSB-1 protein undergoes phase separation in its localization to chromosomes (since its yeast ortholog Rec114 does the same), it is not likely to take on this particular form.
Although the finding of the GFP fusion dependence of droplet formation was not expected, it does not change the main thrust of our research plan which is to find how DSB-1 promotes breaks and how phosphorylation of DSB-1 disables this activity.
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| 今後の研究の推進方策 |
Using the Alphafold structure prediction pipeline, we have found a very well-supported prediction for an interaction between the proteins DSB-1, DSB-2, and DSB-3, as well as between DSB-1 and SPO-11 protein. These interactions are mostly conserved in predictions from other species, from other nematodes to mammals and yeast. Interestingly, however, the C terminus of SPO-11 is distinct in nematodes compared to other organisms, and it is this C terminus that is predicted to interact with DSB-1. We thus expect the possibility of a novel interaction mode between SPO-11 and the Rec114 orthologs to be uncovered in C. elegans.
We will take advantage of the ease of genetic manipulation of C. elegans to create mutations predicted to disrupt (for example, flipping the charge of amino acids predicted to interact ionically) and then restore interactions (by flipping the charge of the interacting residue) between these proteins, to test whether these complexes are made in vivo.
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