Silvia Onesti

Curriculum vitae:
Degree in Chemistry, University of Pavia.
PhD in Biophysics with David Blow and Peter Brick, Imperial College London. 
Postdoctoral Fellow, Biophysics Section, Imperial College London. 
Maître de conférences, Ecole Polytechnique, Palaiseau, Paris. 
Lecturer, Physics Department, Imperial College London. 
Senior Lecturer, Department of Life Sciences, Imperial College London.

Current position:
Head of Structural Biology, Elettra - Sincrotrone Trieste.
Teaching Structural Biology at SISSA.

Contact details:
Address: Sincrotrone Trieste S.C.p.A.
SS 14 - km 163,5 AREA Science Park
34149 Basovizza, Trieste, Italy
Tel: +39-040-3758451
Mobile: +39-366-6878001

Personal webpage

Our research is mainly centered on the structural characterisation of proteins and protein complexes involved in the process of DNA replication and DNA repair in eukaryotic cells. Wherever possible, we will also use the simpler and more stable counterparts present in archaeal organisms.
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. At the same time the DNA needs to be constantly protected and repaired from the damage caused by physical and chemical agents. Although recent genetic and genomic approaches have identified many of the key players in these processes, the detailed analysis of their architecture and the biochemical role of each component is still in progress.

MCM proteins (B. Medagli, S. Jafarkhani, P. De Crescenzio, A. Abdalla Mohammed Khaled)

A key component of the 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 (Costa&Onesti, 2009; Medagli&Onesti, 2013). We have used a combination of electron microscopy and crystallography to understand the architecture of a simplified MCM complex from archaeal cells and have assessed the changes in stoichiometry that the complex undergoes when treated with various substrates (Pape et al., 2003, Costa et al., 2006a&b, Bae et al., 2009; Jenskinson et al., 2009). We have also visualized the initial interaction between MCM and dsDNA, with the DNA wrapping around the N-terminal face of a single hexameric ring and suggested 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, Costa & Onesti, 2008). We are currently tacking the more complex human proteins, and in particular the Mcm8-9 protein complex, involved in recombination and meiosis.

The CMG helicase complex (M. De March, I. Krastanova, B. Medagli, I. Saha)

Although the purified MCM2-7 is not very active, a stable complex comprising Cdc45, MCM2-7 and GINS can be co-purified and has a significant ATP-dependent helicase activity (Onesti&MacNeill, 2013; Medagli et al., 2016). 
Cdc45 is an essential protein conserved in all eukaryotes and is involved in both the initiation of DNA replication and the progression of the replication fork. We detected a weak but significant relationship among eukaryotic Cdc45 proteins and a large family of phosphoesterases that has been described as DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases (Krastanova et al., 2012). Like the RecJ exonucleases, the recombinant human Cdc45 protein is able to bind single-stranded, but not double-stranded DNA. Small angle X-ray scattering data are consistent with a model compatible with the crystallographic structure of the RecJ/DHH family members. 

GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron microscopy (EM), we show that GINS is a compact tetramer in solution as observed in crystal structures, but also forms a double-tetrameric population, detectable by EM (Carroni et al., 2017). This may represent an intermediate step towards the assembly of two replicative helicase complexes at origins, moving in opposite directions within the replication bubble. Reconstruction of the double-tetrameric form, combined with small-angle X-ray scattering data, allows the localisation of the B domain of the Psf1 subunit in the free GINS complex, which was not visible in previous studies and is essential for the formation of a functional replication fork.

RecQ helicases (A. Mojumdar, I. Bagnano, F. Marino, J. Morimoto, M. De March, S. Kenig)

RecQ helicases are essential in the maintenance of genome stability and are highly conserved from bacteria to man. Their importance is clearly demonstrated by the fact that out of the five human RecQ helicases, mutations in three of them cause very serious genetic diseases. In collaboration with Alessandro Vindigni, we are carrying out structural analysis of the human RecQ4 helicase.
We are focussing on various domains of the human RecQ4 helicase. A bioinformatic analysis revealed novel features, inclusing a putative Zn knuckle in the N-terminal region and an RQC domain following the helicase domain (Marino et al., 2013). We carried out a structural and biochemical analysis of the RecQ4 cysteine-rich regions, and showed that it indeed assumes the canonical Zn knuckle fold. We also investigated the effect of a segment located upstream the Zn knuckle that is highly conserved and rich in positively charged and aromatic residues, partially overlapping with the C-terminus of the Sld2-like domain. In both the human and Xenopus proteins, the presence of this region strongly enhances binding to nucleic acids (Marino et al., 2016). We have also expressed and purified the catalytic core of the protein and showed that the putative RQC domain contains two Zn atoms and a number of essential residues. Low resolution structural information obtained by small angle X-ray scattering data suggests that RecQ4 interacts with DNA in a manner similar to RecQ1, whereas the winged helix domain may assume alternative conformations, as seen in the bacterial enzymes. These combined results experimentally confirm the presence of a functional RQC domain in human RecQ4 (Mojumdar et al., 2016; Deka et al., 2017).

PCNA and associate factor PAF15 (Scientist in charge: A. De Biasio; M. De March)

Proliferating Cell Nuclear Antigen (PCNA), the eukaryotic DNA sliding clamp, directs DNA replication and repair by harboring replicative polymerases and other factors. We have recently unveiled the structural basis of the mechanism of sliding of human PCNA on DNA ‒ a helical motion based on short-lived polar interactions ‒ which sheds new light onto previous observations on the function of the PCNA‒polymerase δ holoenzyme (De March et al., 2017). PCNA also interacts with the PCNA-associated factor PAF15, an oncogenic intrinsically disordered protein involved in the regulation of DNA replication and DNA damage bypass (De Biasio et al., 2014; De biasio et al., 2015). We are currently studying the effects of PAF15 on PCNA sliding on DNA, by combining structural studies, molecular dynamics simulations (in collaboration with Ramon Creuhet at the Institute of Advanced Chemistry of Catalonia), and single-molecule methods (in collaboration with Jong-Bong Lee at Postech University). Our long-term goal is to elucidate the mechanistic role of PAF15 in the processes of DNA replication and Translesion Synthesis (TLS), and to gain insight into how PAF15 dysregulation may contribute to cancer development.

Matteo "Jim Watson" De March uses his earphones to illustrate a novel mechanism of sliding of PCNA on DNA based on MD data.

Ciclic nucleotide-gated (CNG) channels (Scientist in charge: L. Napolitano; M. De March)

Cyclic nucleotide-gated (CNG) channels play important roles in transmitting information about vision and smell from sensory cells to the brain, and share a high degree of similarity with K+ channels. Whereas K+ channels discriminate with high accuracy Na+ from K+, CNG channels do not discriminate among different cations. In collaboration with the laboratories of V. Torre and A. Laio (SISSA), we carried out a multidisciplinary study to better understand the specific behaviour of these important membrane proteins. We determined the crystal structure of this protein in complex with a variety of cations  of varying sizes (Li+, Na+, Rb+, Cs+, methylammonium, dimethylammonium). By combining electrophysiology, molecular dynamics simulations and X-ray crystallography we found that the pore region exhibits a dynamic structure and the pore diameter critically depends on the ion within and that a few key residues exhibit large conformational changes. We conclude that the pore of CNG channels is highly flexible and that this flexibility is at the basis of their poor ionic selectivity (Napolitano et al., 2015). 

Selected Publications

(*Corresponding author)

  • Bottega R., Ravera S., Napolitano L.M.R., Chiappetta V., Zini N., Crescenzi B., Arniani S., Faleschini M., Cortone G., Faletra F., Medagli B., Sirchia F., Moretti M., de Lange J., Cappelli E., Mecucci C., Onesti S., Pisani F.M., Savoia A.* (2021). Mitochondrial defect in Warsaw syndrome cells genomic integrity and mitochondrial metabolism defects in Warsaw syndrome cells: A comparison with Fanconi anemia. J Cell Physiol. Online ahead of print.
  • De March M., Onesti S.*, De Biasio A.* (2020) Reply to: "Does PCNA diffusion on DNA follow a rotation-coupled translation mechanism?" Nat. Commun. 11, 4999.
  • Ullah R., Shehzad A., Ali Shah M., De March M., Ismat F., Iqbal M., Onesti S., Rahman M., McPherson M.J.* (2020). C-Terminal Domain of the Human Zinc Transporter hZnT8 Is Structurally Indistinguishable From Its Disease Risk Variant (R325W). Int J Mol Sci, 21, 926.
  • Lazzari E., El-Halawany M., De March M., Valentino F., Cantatore F., Migliore C., Onesti S., Meroni G.* (2019). Analysis of the Zn-binding domains of TRIM32, the E3 ubiquitin ligase mutated in Limb Girdle Muscular Dystrophy 2H. Cells, 8, E254.
  • Bottega R., Napolitano L.M.R., Carbone A., Cappelli E., Corsolini F., Onesti S., Savoia A., Gasparini P., Faletra F.* (2019). Two further patients with Warsaw breakage syndrome. Is a mild phenotype possible? Mol. Genet. Genom. Med. 7, e693.
  • Gonzalez-Magaña A., Ibáñez de Opakua A., Romano-Moreno M., Murciano-Calles J., Merino N., Luque I., Rojas A.L., Onesti S., Blanco F.J., De Biasio A.* (2019). The p12 subunit of human polymerase δ uses an atypical PIP-box for molecular recognition of proliferating cell nuclear antigen (PCNA). J. Biol. Chem. 294, 3947-3956.
  • Pisani F.M.*, Napolitano E., Napolitano L.M.R.Onesti S.* (2018). Molecular and Cellular Functions of the Warsaw Breakage Syndrome DNA Helicase DDX11. Genes, 9, 564. 
  • De March M., Barrera-Vilarmau S., Crespan E., Mentegari E., Merino N., Gonzalez-Magaña A., Romano-Moreno M., Maga G., Crehuet R., Onesti S., Blanco F.J., De Biasio A.* (2018). p15PAF binding to PCNA modulates the DNA sliding surface. Nucleic Acids Res. 46, 9816-9828.
  • Napolitano L.M.R., Marchesi A., Rodriguez A., De March M.Onesti S., Laio A.*, Torre V..* (2018). The permeation mechanism of organic cations through a CNG mimic channel. PLoS Comput. Biol. 14, e1006295.
  • Ali Shah M., Ullah R., De March M., Salahuddin Shaha M., Ismata F., Habib M., Iqbala M., Onesti S., Rahman M.* (2017). Overexpression and characterization of the 100K protein of Fowl adenovirus-4 as an antiviral target. Virus Research    238, 218-225.
  • Deka J., Mojumdar A., Parisse P., Onesti S.* and Casalis L.* (2017). DNA-conjugated gold nanoparticles based colorimetric assay to assess helicase activity: a novel route to screen potential helicase inhibitors. Scientific Rep. 7, 44358.
  • De March M., Merino N., Barrera-Vilarmau S., Crehuet R., Onesti S.*, Blanco F.S.* and De Biasio A.* (2017). Structural basis of human PCNA sliding on DNA. Nat. Commun. 7, 13935.
  • 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). New insights into the GINS complex explain the controversy between existing structural models. Scientific Rep. 7, 40188.
  • Mojumdar A., De March M., Marino F. and Onesti S*. (2017) The human RecQ4 helicase contains a functional RQC domain that is essential for activity. J. Biol. Chem. 292, 4176-4184
  • Ormaza G., Medagli B., Rodríguez J.A., Ibáñez de Opakua A., Merino N., Villate M., Onesti S. and Blanco F.J.* (2016). The tumor suppressor ING4 binds double stranded DNA with micromolar affinity through its disordered central region. FEBS Letters. 591, 425-432.
  • Marino F., Mojumdar A., Zucchelli C. Bhardwaj A., Buratti E., Vindigni A., Musco G. and Onesti S.* (2016). Structural and biochemical characterization of an RNA/DNA binding motif in the N-terminal domain of RecQ4 helicasesScientific Rep. 6, 21501.
  • Medagli B., Di Crescenzio P., De March M. and Onesti S.* (2016). Structure and activity of the Cdc45-Mcm2-7-GINS (CMG) complex, the replication helicase. (Chapter in "The initiation of DNA replication in eukaryotes", Ed. D. Kaplan, Springer).
  • 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). A structural, functional, and computational analysis suggests pore flexibility as the base for the poor selectivity of CNG channels. Proc. Natl. Acad. Sci. USA. E3619-E3628. 
  • Wiedemann C., Ohlenschläger O., Medagli B., Onesti S. and Görlach M. (2013). 1H,  15N and 13C chemical shift assignments for the winged helix domains of two archeal MCM C-termini. Biomol NMR Assign.  
  • Marino F., Vindigni A. and Onesti S.* (2013) Bioinformatic analysis of RecQ4 helicases reveals the presence of a RQC domain and a Zn knuckleBiophys Chem. 177-178, 34-39. 
  • Onesti S. and MacNeill S.A.* (2013) Structure and evolutionary origins of the CMG complex. Chromosoma. 122, 47-53.
  • Medagli B. and Onesti S.* (2013). Structure and mechanism of hexameric helicases. Adv. Exp. Med. Biol. 767, 75-95 (Chapter in "DNA helicases and DNA motor proteins", Ed. M. Spies, Springer).
  • Krastanova I., Sannino V., Amenitsch H., Gileadi O., Pisani F.M. and Onesti S.* (2012). Structural and functional insights into the DNA replication factor Cdc45 reveal an evolutionary relationship to the DHH family of phosphoesterases. J. Biol. Chem. 287, 4121-4128.
  • Costa A. and Onesti S.* (2009). Structural biology of MCM helicases. Crit. Rev. Biochem. Mol. Biol. 44, 326-342.
  • Costa A., Van Dujinen G., Medagli B., Chong J., Sakakibara N., Kelman Z., Nair S.K., Patwardhan A. and Onesti S.* (2008). Cryo-electron microscopy reveals a novel DNA binding site on the MCM helicase. EMBO J. 27, 2250-2258.
  • Costa A., Pape T., van Heel M., Brick P., Patwardhan A. and Onesti S.* (2006). Structural basis of the Methanobacter thermautotrophicus MCM helicase activity. Nucleic Acid Res. 34, 5829-5838.
  • Paraskevopoulou C., Fairhurst S.A., Lowe D.J., Brick P. and Onesti S.* (2006). The Elongator subunit Elp3 contains a Fe4S4 cluster and binds S-adenosylmethionine. Mol. Microbiol. 59, 795-806.
  • Meka H., Werner F., Cordell, S., Onesti S. and Brick P.* (2005). Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II. Nucleic Acid Res. 33, 6435-6444.
  • Pape T., Meka H., Chen S., Vicentini G., van Heel M. and Onesti S.* (2003). Hexameric ring structure of the full-length archaeal MCM complex. EMBO Rep. 4, 1079-1083.
  • Meka H., Daoust G., Bourke-Arnvig K., Werner F., Brick P. and Onesti S.* (2003). Structural and functional homology between the RNAPI subunits A14/A43 and the archaeal RNAP subunits E/F. Nucleic Acid Res. 31, 4391-4400.
  • Todone F., Brick P., Werner, F., Weinzierl R.O.J and Onesti S.* (2001). Structure of an archaeal homologue of the eukaryotic RNA polymerase II RPB4/RPB7 complex. Mol. Cell, 8, 1137-1143.
  • Todone F., Weinzierl R.O.J, Brick P. and Onesti S.* (2000). Crystal structure of RPB5, a universal eukaryotic RNA polymerase subunit and transcription factor interaction target. Proc. Natl. Acad. Sci. USA, 97, 6306-6310.

Ultima modifica il Lunedì, 09 Gennaio 2023 16:05