Elucidation of mechanisms ensuring optimal double-strand break number in meiotic cells

阐明确保减数分裂细胞中最佳双链断裂数的机制

• 批准号: 21H02400
• 负责人: Carlton Peter
• 金额: $ 11.07万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2021
• 资助国家: 日本
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21H02400/
• 关键词: 減数分裂; 線虫; 染色体; DNA二重鎖切断; 二重鎖切断; キナーゼ; ホスファターゼ; リン酸化; 染色体ダイナミクス

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.

在本财政年度，我们继续通过磷酸化调节蛋白质DSB-1来解决减数分裂双链断裂（DSB）控制的机制。我们发现，即使在没有DSB-2蛋白的情况下，将DSB-1中的单个丝氨酸残基突变为丙氨酸，也会导致DSB形成过度活跃。DSB-2的缺失通常会导致由于dsb缺失导致的高胚胎存活率。由于DSB-2的丢失可以很容易地恢复，我们推断它可能是长期基因组稳定所必需的。因此，我们对DSB-1非磷酸化突变系进行了全基因组长读测序，这些突变系已经经历了超过50代的高DSB水平。虽然对该序列信息的分析仍在进行中，但已分离出具有自发突变和自发四倍体化的个体突变动物。由于在野生型动物中从未观察到自发的四倍体化，因此在不可磷酸化的DSB-1突变动物中，基因组变得不那么稳定是合理的。我们试图通过磷酸特异性抗体生产或质谱法直接证明DSB-1的磷酸化尚未成功；然而，我们现在已经完成了flag标记的DSB-1等位基因的构建，具有完全的活力，在western blot上非常干净；该试剂将极大地帮助我们进一步努力表征DSB-1。

项目成果

Nematode chromosomes.

线虫染色体。

• DOI: 10.1093/genetics/iyac014
• 发表时间: 2022-05-05
• 期刊: GENETICS
• 影响因子: 3.3
• 作者: Carlton, Peter M.;Davis, Richard E.;Ahmed, Shawn
• 通讯作者: Ahmed, Shawn

Phosphoregulation of DSB initiation

DSB 引发的磷酸调节

• 发表时间: 2022
• 作者: Noda Taichi;Blaha Andreas;Fujihara Yoshitaka;Gert Krista R.;Emori Chihiro;Deneke Victoria E.;Oura Seiya;Panser Karin;Lu Yonggang;Berent Sara;Kodani Mayo;Cabrera-Quio Luis Enrique;Pauli Andrea;Ikawa Masahito;Peter CARLTON
• 通讯作者: Peter CARLTON