Several genetic and epigenetic alterations are required to drive normal cells toward malignant transformation. Cancer genomes are highly rearranged and characterized by complex chromosomal alterations targeting loci containing cancer-relevant genes. Therefore, mechanisms controlling genome stability are relevant to prevent cancerogenesis. In response to DNA damage, all eukaryotic organisms activate a surveillance mechanism, called DNA damage checkpoint, to arrest cell cycle progression and facilitate DNA repair. Several factors are physically recruited to the damaged sites, and specific kinases phosphorylate multiple targets leading to checkpoint activation. Recent studies have shown that elevated levels of the Polo-like kinase 1 (PLK1) act as a tumour-promoting force possibly by down-regulating the checkpoint response. Notably, studies both in yeast and vertebrates have involved Cdc5/PLK1 in turning off the DNA damage checkpoint and promoting the re-start of cell cycle progression after a DNA damage-induced delay. This finding is attractive not only as a potential explanation for the selective pressure allowing outgrowth of malignant cells with acquired defects in the checkpoint network, but also because it suggests potential therapeutic implications. Indeed, a number of agents inhibiting PLK1 have been developed and are currently evaluated as anticancer drugs. However, the selectivity of these PLK1 inhibitors is low, and off-target effects arising through the inhibition of unrelated kinases or inactivation of essential PLK1 functions in normal cells are serious unwanted consequences of such treatments.
To identify genes and factors that may be more specific targets for anticancer therapies, we have set up a biochemical approach in yeast cells to pull down Polo associated factors. Moreover, the outcomes would be important to further characterize the functional roles of Polo kinases in cells responding to DNA damage. We already started to screen protein extracts prepared from yeast cells responding to DSBs and the pulled down proteins would be identified by mass spectrometry (MS/MS). We will also screen protein extracts prepared from cells treated with specific agents (such as MMS, metyl methanesulfonate, and campthotecine) that cause replication stress.
Budding yeast is an ideal model system to screen and study the functional role of novel factors involved in DNA damage and cell cycle checkpoints. Indeed, it is expected that many of the Polo kinase interactors/substrates identified in yeast may have a counterpart in human cells. Therefore, we aim to apply the same biochemical approach to screen for PLK1 interactors/substrates in human cells. We will produce an affinity column with the PBD of PLK1 purified from E. coli cells. Protein extracts from various human cells lines will be prepared after cells treatment with agents that cause DSBs (ionizing radiations, campthotecin, zeocine) and will be analyzed by the affinity column with the PLK1_PBD. The pulled down proteins will be identified by MS/MS.

During the ongoing screening procedure described above, we will start the biochemical and genetic characterization of those proteins identified both in yeast and in human cells. This part of the project is particularly important to evaluate the possibility that the proteins identified may serve as molecular target for cancer therapy. We will take advantage of yeast model system and our experience in yeast molecular biology to start the preliminary characterization of the PLKs interactors.
We will insert a standard epitope in the corresponding gene to study protein stability, localization and putative post-translational modifications in the presence of an irreparable DSB. We will also produce mutations in the corresponding genes to investigate whether they may affect cellular viability in the presence of DNA damaging agents, possibly affecting the Mec1 signaling pathway. Moreover, by taking advantages of specific genetic and molecular approaches, we will analyze checkpoint adaptation and recovery in the obtained mutant strains.
Depending upon the results in yeast, we will then study the orthologs in human cells of the most interesting factors.
We will design specific siRNA to silence the expression of the corresponding genes and we will test cellular viability and checkpoint activation/inactivation in response to treatment with DNA damaging agents. We will also use a specific genetic system, based upon the conditional expression of the endonuclease Sce1 to study checkpoint response, recovery and adaptation to DSBs in human cells.
We will then study post-translational modifications, protein stability and localization in cells responding to DNA damage of the putative PLK1 targets/interactors we identified. Eventually, to visualize and immunoprecipitate the proteins we will evaluate the possibility to use the expression of etherologous proteins carrying the protein fused with standard epitope cassettes.