DNA-double strand breaks (DSBs) are universally important for initiating meiotic recombination, a key process in the exchange of gene variants within species. The machine that generates these potentially lethal breaks is partly characterized in budding yeast. Its activation is coordinated with DNA replication and requires its tethering to the chromatin. But how is this dangerous breakage machinery turned off?
Research from the groups of Rita Cha (MRC, London) and Franz Klein (MFPL, Vienna) now starts to unveil the mechanisms that turn off the DSB machinery. They have discovered an “OFF button” for the DSB machinery in one of its essential components, the Rec114 protein. In fact, they found three consecutive negative feedback regulations on Rec114, which help to achieve exactly the level of DNA damage that a cell needs for successful meiosis. Together these feedback mechanisms stabilize the DSB levels even under compromised conditions, for instance when the cleavage activity of the DSB machinery is reduced.
Jesus Carballo from the group of Rita Cha decided to investigate if the DSB machinery uses the DNA damage checkpoint to assess whether a break had been made. If so, he reasoned, the checkpoint kinases ATM and ATR might use a component of the DSB machinery as target to turn it off. After identifying ATM/ATR phosphorylation consensus sites in Rec114 by a motif search, Jesus generated a yeast strain with a phospho-mimicking allele (always OFF) and a strain with a non-phosphorylatable allele (always ON). But, while the “always OFF” mutant strain indeed showed a strong reduction in chromosome breakage, he could not demonstrate an increase in the “always ON” mutant.
At this point, Silvia Panizza from the Klein lab could help. Using microarray technology, she could show a genome wide increase in chromosomal breaks in the “always ON” background, and she also found fundamental changes in chromatin binding between the differently phosphorylated Rec114 proteins using an antibody against Rec114 that had been generated in the lab. This led to the conclusion that phosphorylation of Rec114 by ATM and/or ATR inactivates the DSB machinery by limiting its interaction with the DSB hotspots. Two additional, later feedback controls were also revealed: Upon successful local chromosome pairing (one purpose of DSB formation and repair) Rec114 and partners are locally removed from chromatin, while upon completion of chromosome pairing Rec114 is degraded in the whole cell. These two later feedback controls limit the effect of the “always ON” allele.
The Klein lab is very excited about these discoveries and wants to investigate them further. How much does the interception of the communication between DNA hotspots and the DSB machinery at the chromosome axis limit the initiation of recombination – and how much of this limitation is due to the direct regulation of the cleavage activity? The system provides a fascinating example for the important role the chromosome structure plays in the regulation of chromosome metabolism.
Original publication in PLoS Genetics
Carballo JA, Panizza S, Serrentino ME, Johnson AL, Geymonat M, Borde V, Klein F, Cha RS. Budding Yeast ATM/ATR Control Meiotic Double-Strand Break (DSB) Levels by Down-Regulating Rec114, an Essential Component of the DSB-machinery. PLoS Genet. 2013 Jun