Electron Microscopy DNA/Single Molecules

The Electron Microscopy Single Molecule Core Facility supports a variety of research projects by offering high-resolution ad three-dimensional imaging techniques to visualize nucleic acids, proteins and protein-nucleic acid complexes with high resolution. Key applications include:

Structural Analysis of DNA and RNA structures

• Visualization of the in vivo structure of chromosomal DNA and DNA-RNA structures involved in DNA replication, DNA repair, DNA recombination and DNA transcription.
• Investigation of the in vivo structure of structural DNA elements of the chromosomes like repetitive DNA sequences, including centromeres, telomeres, and satellite DNA .
• Structural characterization of episomal DNA elements like extra-chromosomal DNA circles and mitochondrial DNA replication intermediates.

Genome Instability and Cancer Research

• Identification of cancer-associated nucleic acid structures.
• Ultra-structural analysis of DNA, RNA, and DNA-RNA intermediates for potential diagnostic applications.

Proteins and DNA-Protein Complexes

• Visualization of the structure of the proteins and DNA-protein complexes (through negative staining or rotary shadowing). Structure of the protein protofibrils, fibrils and supramolecular protein assembly in normal cells and disease derived cells.
• 2D class averaging and low-resolution 3D reconstruction of protein structure (from negative staining datasets) using the open source CryoSparc (https://cryosparc.com/).

Knowledge Transfer & Network

• Training IFOM researchers in complex single molecule electron microscopy techniques.
• Building a network of skilled users that can apply advanced electron microscopy techniques, methodological innovation.

Future implementations

It is programmed that, in  the next future, the electron microscopy facility DNA/single molecules will acquire an automatic plunge freezer instrument  so that the complete pipe-line  (i.e. automatic plunge freezer,  TALOS F200C G2 transmission microscope, the CetaS/F high sensitive, high S/N camera, the Gatan 626 side entry cryo-transfer sample holder, the EPU and the cryosparc 3D-reconstruction software platforms) to  analyse the 3D structure of the single bio-molecules in their native states in vitreous ice (without staining with heavy metals), will be owned by the IFOM facility and will become available for the IFOM/AIRC research scientists

The facility collaborates closely with IFOM research groups, including those led by Dana Branzei, Vincenzo Costanzo, Fabrizio d’Adda di Fagagna, Ylli Doksani, Marco Foiani, Angela Bachi, and Simona Polo. These partnerships contribute to advancing research on genome integrity and cancer biology, key areas within Athena, IFOM’s ambitious strategic research plan.

La facility EMSM è dotata di strumentazione avanzata per lo studio dell’ultrastruttura e delle interazioni molecolari tramite microscopia elettronica. Include:

• ThermoFisher Transmission electron microscope TALOS F200C G2 
  This is a transmission electron microscope equipped with the high sensitive, high resolution and high signal/noise (S/N) Ceta-S/F camera, the MAPS software, the Tomography software, the EPU software and the inspec3D software. This is a 200KV state of the art transmission electron microscope to study the fine 3D-ultrastructure of tissues, cells and single bio-molecules either at room temperature or in their native states in frozen tissue sections or (for the purified bio-molecules) in vitreous ice.
• Gatan 626 single tilt cryo-transfer sample holder
  (Gatan 655 turbo molecular pumping station, Gatand 626 cryo-transfer-holder with temperature controller)
  The complete cryo-transfer sample holder set-up includes: the Gatan 655 turbo molecular pumping station, the gatan cryo-transfer station and the Gatan 626 cryo-transfer- holder with the temperature controller unit. This is the essential device to load frozen bio-samples into the transmission electron microscope. This sample holder also allows cryo-electron tomography for the 3D reconstruction of tissue structures, cellular structures and single molecules 3D atomic structures in vitreous ice.
• Fischione advanced tomography holder model 2020
  This the essential sample holder necessary for the acquisition of the tilted image series necessary for the 3D reconstruction of tomograms, which are instrumental for the studies of the 3D structure of the bio-samples at atomic resolution (either tissue structures, cells structures or the 3D structure of the bio-molecules) at room temperature.
• Quorum- GloQube plus-glow discharger
  This is the instrument necessary to treat the surface of the electron microscopy supports, which are necessary as support surfaces to apply the bio samples, which will be analyzed with all the high-resolution techniques adopted at the electron microscopy facility
• FEI Tecnai 12 G2 biotwin transmission electron microscope 
  This is a transmission electron microscope with a side mounted Gatan Orius SC-1000 camera. This is an entry-level transmission electron microscope equipped with the open source serial EM software. It allows the acquisition of tomograms at room temperature with a dedicated room temperature tomography sample holder. It offers an extended field of view ideal for quick quality control of bio-sample, quick sample screenings and it is an ideal easy to use platform for the introductory electron microscopy trainings for beginners users from IFOM research groups that would like to start to approach the electron microscopy techniques in a more detailed fashion
• Leica EM ACE600 electron beam (e-beam) metallic coater
  This instrument is used to produce ultra-thin, high-quality metallic layers through the focused electron beam technology on a wide range of electron microcopy supports, ensuring optimal sample preparation for analysis. It is also essential for preparing rotary shadowing samples to visualize nucleic acids. The electron beam configuration (e-beam) is ideal to obtain high quality electron microscopy supports to study the atomic 3D structures of the single bio-molecules
• Leica MED020 electron beam (e-beam) metallic coater
  This instrument is used to produce ultra-thin, high-quality metallic layers (including carbon and platinum layers) on a wide range of electron microscopy  supports, ensuring optimal sample preparation for analysis. It is also essential for preparing rotary shadowing samples to visualize nucleic acids. 

1. Oxidative stress at telomeres triggers internal DNA loops, TRF1 dissociation, and TRF2-dependent R-loops
   Nguyen TT, Mazzucco G, Kyriacou E, Lunardi T, Brandl L, Ahmed W, Doksani Y, Lingner J.
   Nucleic Acids Res. 2025 Apr 10;53(7):gkaf285. doi: 10.1093/nar/gkaf285. PMID: 40219969
2. HMCES corrupts replication fork stability during base excision repair in homologous recombination-deficient cells
   Peña-Gómez MJ, Rodríguez-Martín Y, Del Rio Oliva M, Wijesekara Hanthi Y, Berrada S, Freire R, Masson JY, Reyes JC, Costanzo V, Rosado IV.
   Sci Adv. 2025 Mar 28;11(13):eads3227. doi: 10.1126/sciadv.ads3227. Epub 2025 Mar 26.PMID: 40138423
3. RAD51 protects abasic sites to prevent replication fork breakage
   Hanthi YW, Ramirez-Otero MA, Appleby R, De Antoni A, Joudeh L, Sannino V, Waked S, Ardizzoia A, Barra V, Fachinetti D, Pellegrini L, Costanzo V. Mol Cell. 2024 Aug 22;84(16):3026-3043.e11. doi: 10.1016/j.molcel.2024.07.004. PMID: 39178838 Free article.
4. Unveiling the mechanistic link between extracellular amyloid fibrils, mechano-signaling and YAP activation in cancer.
   Farris F, Elhagh A, Vigorito I, Alongi N, Pisati F, Giannattasio M, Casagrande F, Veghini L, Corbo V, Tripodo C, Di Napoli A, Matafora V, Bachi A. Cell Death Dis. 2024 Jan 11;15(1):28. doi: 10.1038/s41419-024-06424-z. PMID: 38199984 Free PMC article.
5. Sen1 and Rrm3 ensure permissive topological conditions for replication termination
   Ramveer Choudhary, Joanna Niska-Blakie, Mohamood Adhil, Giordano Liberi, Yathish Jagadheesh Achar, Michele Giannattasio, Marco Foiani.
   Cell Rep. 2023 Jul 4;42(7):112747. doi: 10.1016/j.celrep.2023.112747.
6. RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.
   Moore CE, Yalcindag SE, Czeladko H, Ravindranathan R, Wijesekara Hanthi Y, Levy JC, Sannino V, Schindler D, Ciccia A, Costanzo V, Elia AEH.
   J Cell Biol. 2023 May 1;222(5):e202106022. doi: 10.1083/jcb.202106022. Epub 2023 Apr 10. PMID: 37036693
7. The telomerase reverse transcriptase elongates reversed replication forks at telomeric repeats.
   Huda A, Arakawa H, Mazzucco G, Galli M, Petrocelli V, Casola S, Chen L, Doksani Y.
   Sci Adv. 2023 Mar 22;9(12):eadf2011. doi: 10.1126/sciadv.adf2011. Epub 2023 Mar 22. PMID: 36947627 Free PMC article.
8. Short-term molecular consequences of chromosome mis-segregation for genome stability
   Lorenza Garribba , Giuseppina De Feudis , Valentino Martis , Martina Galli , Marie Dumont , Yonatan Eliezer , René Wardenaar , Marica Rosaria Ippolito, Divya Ramalingam Iyer, Andréa E Tijhuis , Diana C J Spierings , Michael Schubert , Silvia Taglietti , Chiara Soriani , Simon Gemble , Renata Basto , Nick Rhind , Floris Foijer , Uri Ben-David , Daniele Fachinetti , Ylli Doksani , Stefano Santaguida
   Nat Commun . 2023 Mar 11;14(1):1353. doi: 10.1038/s41467-023-37095-7.
9. Enrichment of DNA replication intermediates by EdU pull down
   Pessina, F., Romussi, A., Piccini, D., Mazzucco, G, Varasi, M., and Doksani, Y. (2022)
   Methods in Cell Biology. Book Chapter
10. POLq prevents MRE11-NBS1-CtIP dependent fork breakage in the absence of BRCA2/RAD51 by filling lagging strand gaps.
    Anjali Mann, Miguel Angel Ramirez-Otero, Anna De Antoni, Yodhara WijesekaraHanthi, Vincenzo Sannino, Giorgio Baldi, Lucia Falbo, Anna Schrempf, Sara Bernardo, Joanna Loizou, Vincenzo Costanzo.
    Mol Cell 2022. Volume 82, Issue 22,2022,Pages 4218-4231.e8,ISSN 1097-2765, https://doi.org/10.1016/j.molcel.2022.09.013.
11. Rad51-mediated replication of damaged templates relies on monoSUMOylated DDK kinase
    Chinnu Rose Joseph, Sabrina Dusi, Michele Giannattasio, Dana Branzei
    Nat Commun 2022. May 5;13(1):2480. doi: 10.1038/s41467-022-30215-9.
12. Parental histone deposition on the replicated strands promotes error-free DNA damage tolerance and regulates drug resistance
    Valeria Dolce, Sabrina Dusi, Michele Giannattasio, Chinnu Rose Joseph, Marco Fumasoni, Dana Branzei
    Genes Dev 2022. Feb 1;36(3-4):167-179. doi: 10.1101/gad.349207.121. Epub 2022 Feb 3.
13. A rapid method to visualize human mitochondrial DNA replication through rotary shadowing and transmission electron microscopy
    Martin Kosar, Daniele Piccini, Marco Foiani, Michele Giannattasio
    Nucleic Acids Research, Volume 49, Issue 21, 2 December 2021, Page e121, https://doi.org/10.1093/nar/gkab770
14. REV1-Polζ maintains the viability of homologous recombination-deficient cancer cells through mutagenic repair of PRIMPOL-dependent ssDNA gaps
    Angelo Taglialatela, Giuseppe Leuzzi , Vincenzo Sannino , Raquel Cuella-Martin , Jen-Wei Huang , Foon Wu-Baer , Richard Baer , Vincenzo Costanzo , Alberto Ciccia.
    Mol Cell. 2021 Oct 7;81(19):4008-4025.e7. doi: 10.1016/j.molcel.2021.08.016.
15. Checkpoint-mediated DNA polymerase ε exonuclease activity curbing counteracts resection-driven fork collapse.
    Grazia Pellicanò, Mohammed Al Mamun, Dolores Jurado-Santiago , Sara Villa-Hernández , Xingyu Yin , Michele Giannattasio , Michael C Lanz , Marcus B Smolka , Joseph Yeeles , Katsuhiko Shirahige, Miguel García-Díaz , Rodrigo Bermejo
    Mol Cell. 2021 Jul 1;81(13):2778-2792.e4. doi: 10.1016/j.molcel.2021.04.006. Epub 2021 Apr 30
16. Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites.
    Sumedha Agashe , Chinnu Rose Joseph , Teresa Anne Clarisse Reyes , Demis Menolfi , Michele Giannattasio , Anja Waizenegger , Barnabas Szakal , Dana Branzei
    Nat Commun. 2021 Apr 8;12(1):2111. doi: 10.1038/s41467-021-22217-w.
17. The human nucleoporin Tpr protects cells from RNA-mediated replication stress.
    Martin Kosar , Michele Giannattasio , Daniele Piccini , Apolinar Maya-Mendoza , Francisco García-Benítez , Jirina Bartkova , Sonia I Barroso , Hélène Gaillard , Emanuele Martini, Umberto Restuccia , Miguel Angel Ramirez-Otero, Massimiliano Garre, Eleonora Verga, Miguel Andújar-Sánchez , Scott Maynard , Zdenek Hodny , Vincenzo Costanzo , Amit Kumar , Angela Bachi , Andrés Aguilera , Jiri Bartek , Marco Foiani
    Nat Commun.2021 Jun 24;12(1):3937. doi: 10.1038/s41467-021-24224-3.
18. Telomere damage induces internal loops that generate telomeric circles.
    Giulia Mazzucco, Armela Huda, Martina Galli, Daniele Piccini, Michele Giannattasio, Fabio Pessina, Ylli Doksani
    Nat Commun. 2020 Oct 20;11(1):5297. doi: 10.1038/s41467-020-19139-4.
19. Physiological Tolerance to ssDNA Enables Strand Uncoupling during DNA Replication.
    Amaia Ercilla, Jan Benada , Sampath Amitash, Gijs Zonderland, Giorgio Baldi , Kumar Somyajit, Fena Ochs ,Vincenzo Costanzo , Jiri Lukas ,Luis Toledo
    Cell Rep 2020 Feb 18;30(7):2416-2429. doi: 10.1016/j.celrep.2020.01.067
    
20. Dna2 processes behind the fork long ssDNA flaps generated by Pif1 and replication-dependent strand displacement.
    Rossi SE, Foiani M, Giannattasio M.
    Nature Communications. 2018 Nov 16;9(1):4830. doi: 10.1038/s41467-018-07378-5.
21. AND-1 fork protection function prevents fork resection and is essential for proliferation.
    Abe T, Kawasumi R, Giannattasio M, Dusi S, Yoshimoto Y, Miyata K, Umemura K, Hirota K, Branzei D.
    Nature Communications. 2018 Aug 6;9(1):3091. doi: 10.1038/s41467-018-05586-7.
22. Restoration of Replication Fork Stability in BRCA1- and BRCA2-Deficient Cells by Inactivation of SNF2-Family Fork Remodelers.
    Taglialatela A, Alvarez S, Leuzzi G, Sannino V, Ranjha L, Huang JW, Madubata C, Anand R, Levy B, Rabadan R, Cejka P, Costanzo V, Ciccia A.
    Molecular Cell. 2017 Oct 19;68(2):414-430.e8. doi: 10.1016/j.molcel.2017.09.036.
23. Fanconi-Anemia-Associated Mutations Destabilize RAD51 Filaments and Impair Replication Fork Protection.
    Karina Zadorozhny, Vincenzo Sannino, Ondrej Beláň, Jarmila Mlčoušková, Mário Špírek, Vincenzo Costanzo, Lumír Krejčí.
    Cell Rep 2017 Oct 10;21(2):333-340. doi: 10.1016/j.celrep. 2017.09.062.
24. Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments.
    Kolinjivadi AM, Sannino V, De Antoni A, Zadorozhny K, Kilkenny M, Técher H, Baldi G, Shen R, Ciccia A, Pellegrini L, Krejci L, Costanzo V.
    Molecular Cell. 2017 Sep 7;67(5):867-881.e7. doi: 10.1016/j.molcel.2017.07.001. Epub 2017 Jul 27.
25. Xenopus laevis as Model System to Study DNA Damage Response and Replication Fork Stability
    Vincenzo Sannino , Federica Pezzimenti , Stefania Bertora , Vincenzo Costanzo. (2017)
    Methods Enzymol. 2017;591:211-232. doi: 10.1016/bs.mie.2017.03.018. Epub 2017 Apr 8.
26. Centromeric DNA replication reconstitution reveals DNA loops and ATR checkpoint suppression.
    Aze A, Sannino V, Soffientini P, Bachi A, Costanzo V.
    Nature Cellular Biology. 2016 Jun;18(6):684-91. doi: 10.1038/ncb3344. Epub 2016 Apr 25.
27. Rad53-Mediated Regulation of Rrm3 and Pif1 DNA Helicases Contributes to Prevention of Aberrant Fork Transitions under Replication Stress.
    Rossi SE, Ajazi A, Carotenuto W, Foiani M, Giannattasio M.
    Cell Reports. 2015 Oct 6;13(1):80-92. doi: 10.1016/j.celrep.2015.08.073. Epub 2015 Sep 24.
28. Error-free DNA damage tolerance and sister chromatid proximity during DNA replication rely on the Polα/Primase/Ctf4 Complex.
    Fumasoni M, Zwicky K, Vanoli F, Lopes M, Branzei D.
    Molecular Cell. 2015 Mar 5;57(5):812-823. doi: 10.1016/j.molcel.2014.12.038. Epub 2015 Feb 5.
29. Visualization of recombination-mediated damage bypass by template switching.
    Giannattasio M, Zwicky K, Follonier C, Foiani M, Lopes M, Branzei D.
    Nature Structural & Molecular Biology. 2014 Oct;21(10):884-92. doi: 10.1038/nsmb.2888. Epub 2014 Sep 7.