CHARACTERIZATION OF FACTORS INVOLVED IN DNA DAMAGE CHECKPOINT RECOVERY AND ADAPTATION IN YEAST
Creators: name: FERRARI, MATTEO
Publisher: name: Universit� degli Studi di Milano
Year: 2013

description: Genome maintenance and stability are essential goals for all the organisms in order to transfer the correct genetic information to the progeny and to keep fully functional the cellular metabolism. In eukaryotic cells, the presence of DNA lesions causes the activation of an evolutionary conserved mechanism called the DNA damage checkpoint that arrests the cell cycle and stimulates the repair pathways. Double strand breaks (DSBs) are deleterious lesions that can be a serious threat for the cell. In fact, the formation of only one DSB is enough to activate a robust checkpoint response. This DNA lesion is processed by several factors leading to the checkpoint factors recruitment and to the homologous recombination repair. After lesion repair the checkpoint is switched off through a process called recovery; however it has been demonstrated that damaged cells are able to inactivate the checkpoint and restart the cell cycle also in the presence of a persistent DNA lesion, through a checkpoint adaptation process. The reason why this process occurs is not understood, but it has been related to the unrestrained proliferation of cancer cell. In my laboratory we are interested in shedding light on the molecular mechanism of these checkpoint inactivation processes and in the characterization of the involved factors.
During the PhD I focused on the characterization of the functions and regulation of some factors already known to play a role in DSB ends processing and checkpoint switch off: the polo kinase Cdc5, the DNA translocase Tid1/Rdh54 and the nuclease-associated protein Sae2.
First of all we found that high levels of Cdc5 lead to checkpoint switch off and cell cycle re-enter. Relying on this data we decide to perform a biochemical screening in order to identify the Cdc5 targets in presence of DNA damage. This biochemical screening was based on a GST pulldown approach, coupled with tandem mass spectrometry protein identification. As expected, we identify many interactors and among them we found the repair protein Sae2. Interestingly, we found that in presence of elevated levels of Cdc5, Sae2 is hyperphospholylated and binds strongly to the DSB ends. In order to understand the functional role of the Cdc5-Sae2 interaction, I mutagenized different putative Cdc5 binding sites in Sae2. It turned out that Cdc5 binds a C-terminal region of Sae2, which is conserved in other eukaryotes orthologs.
The obtained Sae2 mutants give us interesting results that can be useful for the proper comprehension of the Sae2 function in DNA damage response. Indeed in this thesis I will present preliminary results on the characterization of the Sae2 role in the recovery process.
I was also involved in a project with the aim to study the regulation of Tid1/Rdh54 in the presence of DSB. Tid1 belongs to the Swi2/Snf2 family of chromatin remodellers, is an ATP-dependent DNA translocase able to induce DNA structure remodelling, Rad51 removal from double strand DNA and promote D-loop formation during homologous recombination. Moreover this protein has also a puzzling function in checkpoint inactivation during adaptation since TID1 deletion causes a permanent G2/M block in the presence of one irreparable DSB. I found that Mec1 and Rad53 checkpoint kinases, through a process that requires also the recombination factor Rad51, phosphorylate Tid1 in the presence of DSBs. I also found that Tid1 is recruited on to the DSB site, and that its ATPase activity is dispensable both for the loading and the phosphorylation of the protein. We believe that Tid1 phosphorylation is important to stabilize the binding of the protein on the lesion and to regulate its functional role during checkpoint adaptation.