Investigating phosphoregulation of meiotic recombination using superresolution microscopy

2015 Fiscal Year Annual Research Report

• Project/Area Number: 15H04328
• Research Institution: Kyoto University
• Principal Investigator: Carlton Peter 京都大学, 物質－細胞統合システム拠点, 特定准教授 (20571813)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: 減数分裂 / 線虫 / 組み換え / 超解像度顕微鏡

Outline of Annual Research Achievements

Our previous results showed that the phosphatase PP4 is required for programmed double-strand breaks (DSBs) in C. elegans meiosis. Based on our previous phosphoproteomic screen and known targets of the PP4 phosphatase in mammals and yeast, we hypothesized that activity of the DNA damage kinases ATM or ATR would antagonize PP4's DSB-promoting activity. We have found that mutating the ATR but not the ATM kinase restores DSB formation in PP4 mutants, suggesting that PP4 and ATR have opposing effects on DSB formation. Loss of PP4 function also rescues DNA replication errors seen in single ATR mutants. Taken together, our research results suggest strongly that PP4 and ATR play balancing roles in creating the correct number of DSBs to promote timely crossover recombination.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

We have taken significant steps toward identifying PP4-interacting partners important for correct meiotic recombination. Now that we also know ATR is a genetically counteracting kinase for PP4, we have a new route toward molecular mechanisms by investigating proteins that are hyperphosphorylated in PP4 mutants, but hypophosphorylated in ATR mutants. We have several such candidate proteins under current investigation as a result of our experiments in FY2015.

Strategy for Future Research Activity

We will investigate the functional phosphorylation two known accessory factors to SPO-11, the enzyme responsible for DSB formation, as they both contain ATR consensus phosphorylation sites ([ST]Q). We will test a model wherein initial DSB formation causes local hyperphosphorylation of these factors by ATR, thus preventing further DSB formation. If the factors under consideration are not targets of PP4, we will inhibit other phosphatases such as PP1 and PP2A, to determine if other phosphatases are responsible for positive regulation of DSBs.
We will also discover additional targets of the PP4 phosphatase, using a fusion between the PP4 catalytic subunit and bacterial biotin ligase (proximity-based ligation of biotin).