| 研究実績の概要 |
Building on the results published in 2022, we continued to assess the dual functions of DSB-1 in promoting and inhibiting meiotic DNA double-strand breaks.
Our live imaging experiments with EGFP-DSB-1 revealed a droplet-like localization in early prophase nuclei, shifting to a chromosome axis localization in later prophase. We were concerned that the droplet could be the result of the dimerization of EGFP. We therefore mutated EGFP in situ to the monomeric form (mEGFP), and this showed the same patterns of localization compared to non-monomeric EGFP-DSB-1. To gain further confidence, we constructed another fusion, mScarlet-DSB-1. This localization was extremely dim in early prophase, making assessment difficult; however, in later prophase, we saw the same axial localization as EGFP-DSB-1. In summary, we have good evidence for axial localization of DSB-1, but confirmation of the droplet will require further protein fusions.
Next, to assess whether the dephosphorylation of DSB-1 by PP4 requires binding of PP4 to the FXXP domains of DSB-1, we mutated the two FXXP domains closest to the key phosphoserine residue S186 (changing F and P residues to A). We hypothesized that if these sites were necessary, then PP4 would not be able to bind to and dephosphorylate DSB-1, and so would expect to see a similar effect to removing PP4; that is, a loss of double-strand breaks. However, we saw no change in break levels in these mutants. DSB-1 has 5 FXXP sites, and so our next strategy is to mutate the other 3 sites in combination.
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