Sannino Vincenzo

I work as post-doc at the FIRC Institute of Molecular Oncology (IFOM, Milan - Italy) in the laboratory of Prof. Vincenzo Costanzo. I am currently working on a project aimed at the identification of uncharacterized DNA repair pathways activated in the absence of the tumor suppressor BRCA2. My approach is based on a starting proteomic screening carried out with the Xenopus system and a second validation phase carried out with both Xenopus extracts and mammalian cells. With Prof. Costanzo I previously collaborated on a project aimed at describing DNA replication at specific chromosomal regions such as centromeres. Using bacterial artificial chromosomes and the Xenopus laevis egg extract system we were able to characterize the chromatin assembly and replication dynamics of centromeric alpha-satellite DNA. By using mass spectrometry together with electron microscopy analyses we managed to describe the assembly of a specific protein matrix and the formation of Topoisomerase I-dependent DNA loops. This peculiar conformation has been shown to switch off the ATR-dependent checkpoint by preventing RPA hyper-loading thus facilitating the replication of centromeric DNA. I personally carried out the electron microscopy analyses of the DNA molecules for this paper. I succeeded in setting up a method to directly visualize the centromeric DNA chromatin structure for the first time. Moreover, a successful collaboration has been carried out with Dr. Luca Pellegrini (Cambridge University, Cambridge, UK). This allowed me to continue the work previously done with Dr. Francesca Pisani at IBP-CNR to describe the Cdc45 DNA replication factor structure and function. By exploiting the structural data obtained by X-ray crystallography we managed to finely map regions of Cdc45 involved in the interaction with the other components of the CMG (Cdc45-Mcm2-7-GINS) complex, the eukaryotic replicative DNA helicase. Specific mutants of Cdc45 in those regions were functionally analyzed using the Xenopus egg extract system and DNA replication dynamics were described in the absence of a properly-assembled CMG complex. I personally carried out all the functional analyses with the Xenopus system. In 2013 I got a PhD in Biotechnological Sciences and in the last 12 years I have been working in the biochemistry/molecular biology field being interested mostly in the characterization of proteins and enzymes involved in the DNA replication mechanism. Especially during the work carried out at the Institute of Protein Biochemistry (IBP - CNR), under the supervision of Dr. Francesca Pisani, I had the opportunity to become very familiar with the production, purification and characterization of recombinant proteins produced in bacteria as well as in human and insect cells. One of the main goal that I reached collaborating with Dr. Pisani at IBP was to reconstitute an active recombinant CMG complex produced and purified from insect cells. The CMG complex is actually the main replicative DNA helicase and it is composed by 11 different proteins that were needed to be co-expressed in insect cells in order to purify a stable and functional CMG complex. To this purpose we exploited a new cloning system in order to combine the 11 ORFs (Open Reading Frames) coding for the 11 different proteins on 3 viral vectors. I am expert in the mostly employed affinity chromatography techniques as well as in the use of FPLC systems. During my lab experience I had the opporunity to experiment several kind of enzymatic assays and I am able to devise new ones. I am very expert in biochemistry, use of Xenopus laevis as a model system to study DNA replication and DNA damage response and use of Transmission Electron Microscopy for analysis of DNA replication intermediates starting from X. laevis extract, X. laevis embryos and mammalian cells. I am very well capable of carrying out independent research work as well as organizing team work, I have strong motivation and a constant determination to hard work.