Head of Structural Biology
Master Degree in Chemistry, University of Pavia
PhD in Structural Biology, Department of Physics, Imperial College London
Post-doctoral fellow in Prof. David Blow’s group at Imperial College.
CNR Research Scientist, Department of Genetics, University of Pavia.
Lecturer, Department of Physics, Imperial College.
Senior Lecturer, Department of Biological Sciences, Imperial College.
Teaching Structural Biology at the International School of Advanced Studies (ISAS/SISSA), within the PhD Program in Structural and Functional Genomics http://www.sissa.it/phdgenomics/index.php/about-us
Structural biology is an interdisciplinary research area, requiring expertises from both the life sciences and the physical sciences. We apply molecular and structural biology tools to study the basic genetic processes within the cell, such as DNA replication and transcription. We use protein crystallography to determine the atomic structure of eukaryotic and archaeal proteins involved in these processes. Crystallographic studies are complemented by the concomitant use of electron microscopy to visualise the architecture of large complexes.
DNA replication and transcription are crucial event in the cell cycle, underpinning cellular processes with important consequences such as cell proliferation and genome stability. Failure to control these processes causes chromosome instability, which can lead to the development of cellular abnormalities, genetic disease and the onset of cancer.
RNA polymerases (RNAP) are complex enzymes that contain a large catalytic core conserved from prokaryotes to human. In addition to this conserved unit, archaeal and eukaryotic polymerases also include a number of smaller polypeptides, most of which are absolutely required for transcription. We investigate the 3D structures of various subunits in order to learn more about RNA polymerase architecture. We have determined the crystal structure of S. cerevisiae RPB5, one of the subunits shared by all three eukaryotic RNAPs (Todone et al., 2000) and the the complex between the Methanococcus jannaschii, the archaeal homologues of Rpb7 and Rpb4 (Todone et al., 2001) and proposed a function for Rpb7 in binding the nascent RNA transcript. The structure suggested an intriguing and previously undetected homology between the RNA polymerase II Rpb4/Rpb7 complex and RNA polymerase I subunits A14 and A43 (Meka et al., 2003). We have also determined the structure of the human Rpb4/Rpb7 heterodimer and complemented the structural analysis with biochemical studies directed at dissecting the RNA binding properties of the complex (Meka et al., 2005).
Although our understanding of eukaryotic DNA replication has improved considerably in recent years, the detailed mechanism of initiation of the reaction is still not known.
MCM proteins are large macromolecular assemblies acting as the replicative helicases and playing a key role in the initiation of DNA replication. We have obtained an initial three-dimensional reconstruction from negatively stained MCM particles from M. thermoautotrophicum (Pape et al., 2003). We have assessed the changes in stoichiometry that the complex undergoes when treated with various substrates (Costa et al., 2006a). 3D reconstructions were carried out for a dsDNA treated and an ADP.AlFx treated sample, respectively assembling as double hexamer and double heptamer (Costa et al., 2006b). The electron density maps display an unexpected asymmetry, between the two rings, suggesting that large conformational changes can occur within the complex. We have visualized a novel interaction between MCM and dsDNA, with the DNA wrapping around the N-terminal face of a single hexameric ring. This interaction requires a conformational change within the outer belt of the MCM N-terminal domain, exposing a previously unrecognized helix-turn-helix DNA-binding motif. We suggest that this represents an initial site of interaction, prior to the loading and activation of the complex to function as a helicase at the fork (Costa et al., 2008).
We have expressed and purified in recombinant form the human replication factor Cdc45, and carried out biochemical and structural studies. We detected a weak but significant relationship among eukaryotic Cdc45 proteins and the RecJ ssDNA exonucleases. Small angle X-ray scattering data are consistent with a model compatible with the crystallographic structure of the RecJ/DHH family members (Krastanova et al., 2012).
Member of the Editorial Board of Scientific Reports, a new open access publication from Nature Publishing Group, covering all areas of natural sciences (http://www.nature.com/srep/about/index.html).
Chairman of the of INSTRUCT Italian Working Group on Complementary Techniques (http://www.cerm.unifi.it/about-cerm/italian-users-of-instruct).
Member of the IUCr Commission on Biological Macromolecules (http://www.iucr.org/iucr/commissions/cbm.html)
Member of the International Programme Committee for the organization of the IUCR 23rd Congress and General Assembly to be held in Montreal 5-12 August 2014. (http://iucr2014.org/side_organization/international_program_committee_e.shtml)
Member of the Programme Committee for the organization of the Meeting 2014: Crystal (cl-)Year to celebrate the International Year of Crystallography to be held in Turin 16-17 October 2014.
Member of the Programme Committee for the organization of the 29th European Crystallographic Meeting (ECM29) to be held in Rovinj 23-28 August 2015.
Associazione Italiana per la Ricerca sul Cancro (AIRC: http://www.airc.it/)
Programma INTERREG Crossborder Cooperation Programme Italy-Slovenia 2007-2013.
French Muscular Dystrophy Association (AFM Téléthon)
Sincrotrone Trieste S.C.p.A., SS 14 - km 163,5 - AREA Science Park, 34149 Basovizza, Trieste ITALY
Email: firstname.lastname@example.org, Tel. +39 040 3758451, Mob +39 366 6878001
DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors. Deka J., Mojumdar A., Parisse P., Onesti S.* and Casalis L.* (2017). Scientific Rep. In press.
Structural basis of human PCNA sliding on DNA. De March M., Merino N., Barrera-Vilarmau S., Crehuet R., Onesti S*., Blanco F.S*. and De Biasio A.* (2017). Nat. Commun. 7, 13935.
New insights into the GINS complex explain the controversy between existing structural models. Carroni M., De March M., Medagli B., Krastanova I., Taylor I.A., Amenitsch H., Araki H., Pisani F.M., Patwardhan A. and Onesti S.* (2017). Scientific Rep. In press.
The human RecQ4 helicase contains a functional RQC domain that is essential for activity. Mojumdar A., De March M., Marino F. and Onesti S.* (2016). J. Biol. Chem. [Epub ahead of print]
The tumor suppressor ING4 binds double stranded DNA with micromolar affinity through its disordered central region. Ormaza G., Medagli B., Rodríguez J.A., Ibáñez de Opakua A., Merino N., Villate M., Onesti S. and Blanco F.J.* (2016). FEBS Letters. [Epub ahead of print]
Structure and activity of the Cdc45-Mcm2-7-GINS (CMG) complex, the replication helicase. Medagli B., Di Crescenzio P., De March M. and Onesti S.* (2016). (Chapter in The initiation of DNA replication in eukaryotes, Ed. D. Kaplan, Springer).
Structural and biochemical characterization of an RNA/DNA binding motif in the N-terminal domain of RecQ4 helicases. Marino F., Mojumdar A., Zucchelli C. Bhardwaj A., Buratti E., Vindigni A., Musco G. and Onesti S.* (2016). Scientific Rep. 6, 21501.
Status of the crystallography beamlines at Elettra. Lausi A.*, Polentarutti M., Onesti S., Plaisier J.R., Busetto E., Bais G., Barba L., Cassetta A., Campi G., Lamba D., Pifferi A., Mande S.C., Sarma D.D., Sharma S.M., Paolucci G. (2015). Eur. Phys. J. Plus 130, 43-51.
A structural, functional, and computational analysis suggests pore flexibility as the base for the poor selectivity of CNG channels. Napolitano L.M.R., Bisha I., De March M., Marchesi A., Arcangeletti M., Demitri N., Mazzolini M., Rodriguez A., Magistrato A., Onesti S.*, Laio A.* and Torre V.* (2015). Proc. Natl. Acad. Sci. USA 112, E3619-E3628.
Bioinformatic analysis of RecQ4 helicases reveals the presence of a RQC domain and a Zn knuckle. Marino F., Vindigni A. and Onesti S.* (2013). Biophys Chem. 177-178, 34-39.
Structure and evolutionary origins of the CMG complex. Onesti S. and MacNeill S.A.* (2013). Chromosoma 122, 47-53.
Structural and functional insights into the DNA replication factor Cdc45 reveal an evolutionary relationship to the DHH family of phosphoesterases. Krastanova I., Sannino V., Amenitsch H., Gileadi O., Pisani F.M., Onesti S.* (2012). J. Biol. Chem. 287, 4121-4128.
Structural biology of MCM helicases. Costa A. and Onesti S.* (2009). Crit. Rev. Biochem. Mol. Biol. 44, 326-342.
Cryo-electron microscopy reveals a novel DNA binding site on the MCM helicase. Costa A., Van Dujinen G., Medagli B., Chong J., Sakakibara N., Kelman Z., Nair S.K., Patwardhan A. and Onesti S.* (2008). EMBO J. 27, 2250-2258.
Structural basis of the Methanobacter thermautotrophicus MCM helicase activity. Costa A., Pape T., van Heel M., Brick P., Patwardhan A. and Onesti S.* (2006) Nucleic Acid Res. 34, 5829-5838.
The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine. Paraskevopoulou C., Fairhurst S.A., Lowe D.J., Brick P. and Onesti S.* (2006) Mol. Microbiol. 59, 795-806.
Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II. Meka H., Werner F., Cordell, S., Onesti S. and Brick P.* (2005) Nucleic Acid Res. 33, 6435-6444.
Hexameric ring structure of the full-length archaeal MCM complex. Pape T., Meka H., Chen S., Vicentini G., van Heel M. and Onesti S.* (2003) EMBO Rep. 4, 1079-1083.
Structural and functional homology between the RNAPI subunits A14/A43 and the archaeal RNAP subunits E/F. Meka H., Daoust G., Bourke-Arnvig K., Werner F., Brick P. and Onesti S.* (2003) Nucleic Acid Res. 31, 4391-4400.
Structure of an archaeal homologue of the eukaryotic RNA polymerase II RPB4/RPB7 complex. Todone F., Brick P., Werner, F., Weinzierl R.O.J and Onesti S.* (2001) Mol. Cell, 8, 1137-1143.
Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target. Todone F., Weinzierl R.O.J, Brick P. and Onesti S.* (2000) Proc. Natl. Acad. Sci. USA, 97, 6306-6310.