Our genome is constantly experiencing DNA damage. As damage accumulates, our cells respond and stop the process of cell division, giving time for repair mechanisms to take action. To do this, two major protein kinases, known as ATR and ATM, encoded by the well-known tumor suppressor gene, can activate a series of phosphorylation events. The downstream reads cascaded by these checkpoints have been well described, but the regulation of the major kinases themselves remains a mystery. Nicole Hustedt, a student at Susan Gasser Laboratory, examined the problem using a combination of complex yeast genetics and phosphoproteomics. She found that yeast ATR homologue, a key regulator of yeast Mec1 kinase, is a phosphatase that directly reverses the repair of many damage-induced Mec1 kinase targets. The identified phosphatase, known as PP4, not only dephosphorylates Mec1 targets, but also directly interacts with kinase to regulate its activity in cell cycle.

         This points to a remarkable regulatory principle: the main kinase ATR/Mec1 seems to be counteracted by a phosphatase, which forms a complex with the enzyme. This chimerism can regulate DNA damage checkpoint response to protect cells from DNA damage.

         Appropriate replication of genome is a critical process for organism survival. However, excessive accumulation of errors and injuries in our chromosomes can lead to cell death or cancer. To ensure genomic integrity, checkpoint kinases control cell division cycles through a series of phosphorylation events. These can delay cell division and stimulate appropriate and timely repair.

         In order to reveal the regulation mechanism of DNA damage checkpoint kinase, Susan Gasser of Friedrich Miescher Institute of Biomedical Sciences and his team used powerful genetic techniques that have been optimized in budding yeast, called high-throughput genetic interaction mapping (E-MAP). The combination of synthetic genetics and phosphorylation interaction with proteomics was carried out on the protein analysis device of FMI. As a result, researchers found a new regulatory interaction that controls DNA damage checkpoints during genome replication.

         Large combinatorial genetic method E-MAP allows researchers to identify a pair of genes that act together or antagonize each other in a given process. This is applied to Mec1-regulated DNA damage response. Nicole Hustedt, a FMI graduate student, found that only one pair of genes could effectively offset defects in Mec1-mediated checkpoint cascades. The two genes found encode PP4 subunit, a phosphatase. Relevant research results published in the recent Journal of Molecular Cell indicate that PP4 phosphatase Pph3-Psy2 can regulate a large number of Mec1 phosphorylation targets. Therefore, it contributes to the balance between phosphorylation and dephosphorylation. This allows for a rapid and effective response to injury-induced checkpoint activation, ultimately restoring cell cycle. Interestingly, they also found that PP4 phosphatase could physically interact with Mec1 to form a complex containing a kinase and a phosphatase. 

          Gasser commented: "These two enzymes act in a coordinated but opposite manner, which makes it easy to regulate Mec1 activity in the cell cycle. Neutralization modification is the Yin and Yang sides of DNA damage checkpoint. The mechanisms that control cell cycle and activate checkpoints are conservative between organisms. Mec1 and Pph3-Psy2 have mammalian homologues, and they also interact. Gasser commented: "By understanding this interaction in yeast, we can jump to human cells, suggesting that the regulation of ATR in mammalian Mec1 homologues uses the same mechanism. This helps us understand some processes that ensure genomic integrity and thus prevent carcinogenic transformation. 

         Epistatic miniarray analysis: E-MAP

         Using E-MAP, scientists are looking for mutant proteins that can either mitigate or aggravate the effects of another known mutation. Such gene interactions are called strong or additional, respectively, and their analysis can reveal the structure and function of pathways. E-MAP can measure the genetic interaction of proteins in a high-throughput and semi-quantitative way. The experiment is based on a quantitative phenotype, such as growth. Then, the method identifies mutation combinations that are better or worse in some cases. The data are then clustered so that researchers can identify genes that interact directly or indirectly with the first mutation. In the experiments mentioned above, scientists measured mutant growth in the absence of functional Mec1. Thus, the proteins shielding the effect of Mec1 mutation were identified.