Silvia Onesti
Silvia Onesti
affiliation: Sincrotrone Trieste S.C.p.A.
research area(s): Molecular Biology, Cancer Biology
Course: Molecular Biomedicine
University/Istitution: Università di Trieste
Higher Education
1982-1987: Laurea in Chimica (Chemistry degree), University of Pavia. Alumnus of Collegio Ghislieri (Pavia).
1987-1991: PhD in Biophysics, Imperial College London, Physics Department, under the supervision of Peter Brick & David Blow.
1991-1994: Post-doctoral research fellow in Prof. David Blow’s group at Imperial College London.
1994-1995: CNR Research Scientist, University of Pavia.
1995-2004: Lecturer, Department of Physics, Imperial College London.
2004-2008: Senior Lecturer, Department of Biological Sciences, Imperial College London.
2008-present: Head of Structural Biology, Sincrotrone Trieste
2008-present: Teaching Structural Biology, International School for Advanced studies (SISSA)
Member of the Editorial Board of Scientific Reports, a new open access publication from Nature Publishing Group, covering all areas of natural sciences.
Chairman of the of INSTRUCT Italian Working Group on Complementary Techniques.
Member of the Commission on Biological Macromolecules of the International Union of Crystallography.
Our research is mainly centered on the structural characterisation of proteins and protein complexes involved in the process of DNA replication in eukaryotic cells. Wherever possible, we will also use the simpler and more stable counterparts present in archaeal organisms.
Structural biology is an interdisciplinary research area, requiring expertises from both the life sciences and the physical sciences. We have recently set-up a new Structural Biology Laboratory at the italian synchrotron facility Elettra, including state-of-the art facilities for high-throughput cloning, large-scale expression, purification of recombinant proteins and crystallization. We use protein crystallography to determine the atomic structure of these proteins, as well as biochemical and biophysical approaches to understand how they work. Crystallographic studies are complemented by the concomitant use of electron microscopy to visualise the architecture of large complexes and/or small-angle X-ray scattering (SAXS) to obtain additional structural information.
Eukaryotic DNA replication is a highly coordinated and tightly regulated process. Due to the large genome size, eukaryotic cells initiates DNA replication at multiple origins and complex networks of proteins, under strict cell-cycle control, are required to ensure that each origin is used only once and no segment of DNA is left un-replicated or undergoes multiple rounds of replication. Although recent genetic and genomic approaches have identified many of the key players, an understanding of their architecture and biochemical role of each component at the replication fork is still lacking.
These are crucial events 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 diseases and the onset of cancer.
Paraskevopoulou, C., Fairhurst, S.A., Lowe, D.J., Brick, P. & Onesti, S. (2006). The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine. Mol. Microbiol. 59, 795-806.
Palmieri, G., Casbarra, A., Fiume, I., Catara, G., Capasso, A., Marino, G., Onesti, S. & Rossi, M. (2006) Identification of the first archaeal oligopeptide binding protein from the hyperthermophile Aereopyrum pernix. Extremophiles, 10, 393-402.
Costa A., Pape T., van Heel M., Brick P., Patwardhan A. & Onesti S. (2006) Structural studies of the archaeal MCM complex in different functional states. J. Struct. Biol., 156, 210-219.
Costa A., Pape T., van Heel M., Brick P., Patwardhan A. & Onesti S. (2006). Structural basis of the Methanobacter thermautotrophicus MCM helicase activity. Nucleic Acid Res. 34, 5829-5838.
Vinaya Sampath V., Bindu Balakrishnan B., Verma-Gaur J., Onesti S. & Sadhale P.P. (2008) Unstructured N terminus of Rpb4 contributes to the interaction of Rpb4/Rpb7 subcomplex with the core RNA polymerase II in S. cerevisiae. J. Biol. Chem., 283, 3923-3931.
Costa A & Onesti, S. (2008). The MCM complex: (just) a replicative helicase? Biochem. Trans. 36, 136-140.
Costa A., van Dujnen G., Medagli B., Chong J., Sakakibara N., Kelman Z., Nair S.K., Patwardhan A. & Onesti S. (2008). Cryo-electron microscopy reveals a novel DNA binding site on the MCM helicase. EMBO J., 27, 2250-2258.
Bae B., Chen Y.-H., Costa A., Onesti S., Brunzelle J.S., Lin Y., Cann I.K.O. & Nair S.K. (2009) Crystal Structure of an Archaeal MCM Homolog Provides Insights into the Architecture of the Replicative Helicase. Structure 17, 211-222.
Jenkinson, E.R., Costa A., Leech, A.P., Patwardhan A., Onesti S. & Chong, J.P (2009). Mutations in sub-domain B of the MCM helicase affect DNA binding and modulate conformational transitions. J. Biol. Chem. 284, 5654-5661.
Costa, A & Onesti, S. (2009). Structural biology of MCM helicases. Crit. Rev. Biochem. Mol. Biol. 44, 326-342.
Project Title:
Structural and functional studies of human replication factors
Due to their large size of their genomes, eukaryotic cells need to start DNA replication from multiple sites, requiring sophisticated control mechanisms that ensures that all DNA is copied once and only once per cell cycle. A large number of protein participate to this complex orchestrated process. We are carrying out structural and functional studies on some of these: we can express and purify to homogeneity some human replication factors, including Cdc45, the GINS complex, the primase heterodimer and the helicases RecQ1 and RecQ4. These are all essential factors having essential roles in the process that leads to the “firing” of replication origins, the establishment and progression of the replication fork, as well as genome stability. All of these proteins are only present in proliferating cells, and are therefore under study either as tumor biomarkers or cancer drug targets.

Project Title:
Elucidation of the structure and function of human MCM helicases
A key component of the replication fork is the replicative helicase, opening up the DNA double helix ahead of the fork movement. All eukaryotic organisms possess six homologous MCM proteins (MCM2-7) that form hetero-hexamers. We are carrying out a detailed structural and functional analysis of human MCM complexes in order to determine the crystal structures of some of these proteins. We will also use biochemical tools to establish the functional properties of fragments and subdomains. Although our primary target will be structure determination using X-ray diffraction, we will exploit other techniques, such as electron microscopy and/or small-angle X-ray scattering to obtain a low resolution picture of large assemblies that may be difficult to tackle by protein crystallography. MCM proteins are present only in proliferating cells and are highly expressed in malignant human cancers cells and pre-cancerous cells undergoing malignant transformation and are therefore ideal diagnostic biomarkers for cancer and possibly targets for anti-cancer drug development.