Smith, Brian O.

Spies, Maria

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     Spies, M. and Smith, B. O.  (2017) Protein-nucleic acids interactions: new ways of connecting structure, dynamics and function. Biophysical Reviews, 9(4), pp. 289-291. (doi:10.1007/s12551-017-0284-4)  This is the author’s final accepted version.  There may be differences between this version and the published version. You are advised to consult the publisher’s version if you wish to cite from it.  http://eprints.gla.ac.uk/145910/                            Deposited on: 22 August 2017         Enlighten – Research publications by members of the University of Glasgow http://eprints.gla.ac.uk   Protein-nucleic acids interactions: new ways of connecting structure, dynamics and function Maria G. Spies1 & Brian O. Smith2 1Carver College of Medicine, University of Iowa, 51 Newton Rd., 4-532 BSB, Iowa City, IA 52242, USA 2Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary & Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK.  Molecular machines that act on nucleic acids, DNA and RNA, are at the heart of the cellular information processing. A coherent description of the interactions involved in their assembly, activities and regulation affords a quantitative understanding of how transcription factors and DNA repair proteins find their unique targets among millions of nonspecific sequences and undamaged DNA bases, how the intricate choreography of DNA replication, recombination and repair, and gene expression is regulated, how viral particles self-assemble and how chromosomes are organized inside living cells. These important questions are not easy to answer. This is because transactions between proteins and nucleic acids commonly involve extended surfaces with multiple interaction epitopes, the resulting macromolecular assemblies are non-homogenous and dynamic; and the structures of multicomponent protein-DNA and protein-RNA complexes are often refractory to analysis by traditional X-ray crystallography and nuclear magnetic resonance (NMR).  New techniques and clever adaptations and combinations of the state-of-the-art approaches are therefore needed.    Our selection of speakers cover exciting new developments, both technological and conceptual, in determining the structures of protein-DNA and protein-RNA complexes, as well as in connecting the structure, dynamics and activity of these complexes using methods of single-molecule biophysics. While understanding of nucleoprotein architecture at atomic resolution and how it changes in response to regulatory events can be achieved using structural biology techniques, single-molecule techniques allow us to visualize and interrogate biologically important molecules individually, in real time. For both structural biology and single molecule techniques, the ability to validate observations made in vitro under physiological conditions is both more important and more feasible (Sakakibara, Sasaki et al. 2009, Ito, Mikawa et al. 2012, Hamatsu, O'Donovan et al. 2013). The ability to observe and directly manipulate individual protein and nucleic acid molecules provides further unprecedented insight into the kinetics of the molecular recognition steps, which are often obscured in bulk biochemical and biophysical studies (Cornish and Ha 2007, Boehm, Subramanyam et al. 2016). During the last decade or so, technological advances in structural biology and single-molecule biophysics have brought new vibrancy and excitement to the field of nucleoprotein interactions, which we hope we capture in the selection of this session's topics.  The three invited talks and the three oral presentations selected for this session exemplify the breadth of excellence at the frontiers of structural and single-molecule research: The ParB protein of Bacillus subtilis is the subject of Fernando Moreno-Herrero's talk. Fernando Moreno-Herrero (Centro Nacional de Biotecnologia, Spain) has been leading the field in atomic force microscopy (AFM) studies of protein-nucleic acid interactions (Fuentes-Perez, Dillingham et al. 2013). ParB condenses and bridges DNA molecules in the context of bacterial chromosome organisation and dynamics which the Moreno-Herrero lab investigates in collaboration with the Dillingham (Univ Bristol), Crump (Univ. Bristol), Sobbot (Univ. Atwerp) and Murray (Univ. Newcastle) groups. In his talk he shows that the C-terminal domain of B. subtillis ParB is critical for dynamic DNA binding and bridging using magnetic tweezers (MT) and a combined MT - total internal reflection microscopy (TIRF) setup (Taylor, Pastrana et al. 2015). The CRISPR-Cas system is one of the hottest topics in biology currently due to its rapid adoption as the genome editing tool of choice, but there is still plenty to discover about its mechanism and this knowledge will inform the development of more faithful and less promiscuous tools (Spies 2014). Ralf Seidel (Leipzig University, Germany) describes work using MT (Daldrop, Brutzer et al. 2015) to interrogate the mechanism of target recognition by CRISPR-Cas system. By applying force and torque to the ends of the DNA molecule his group were able to probe the binding affinity of CASCADE for DNA targets with differently positioned imperfections (Szczelkun, Tikhomirova et al. 2014, Rutkauskas, Krivoy et al. 2017).  Metabolic disorders that originate from mitochondrial defects have been the subject of much public interest in recent years and Hanna Yuan (Academia Sinica, Taiwan) presents her group's latest work on Endonuclease G (EndoG), which digests paternal mitochondrial DNA during embryogenesis. EndoG also has a role in mature mitochondria integrating DNA replication and repair, apoptosis and response to oxidative stress. Using X-ray crystallography and studies in C. elegans, the Yuan group reveal how EndoG degrades DNA without sequence specificity and shown how EndoG acts as a ROS sensor presenting new avenues for the prevention and treatment of the diseases that result from oxidative stresses (Lin, Wu et al. 2016, Zhou, Li et al. 2016). Electron Paramagnetic Resonance (EPR) has recently had a renaissance in biomolecular structural biology and Christoph Gmeiner (ETH Zurich, Switzerland) describes how the long-range distance restraints produced by combining site-directed spin labelling with pulsed EPR can be used in conjunction with short-range NMR restrains to determine the 3D structure of the PTBP1/EMCV protein-RNA complex (Duss, Yulikov et al. 2014, Duss, Yulikov et al. 2015).  NMR spectroscopy remains the only technique that can simultaneously provide detailed structural and dynamic information on biomolecules in solution and Bruno Kieffer (IGBMC, France) presents work that reveals the complex allosteric mechanism by which post-translational phosphorylation regulates the retinoic acid receptor's recognition of target DNA allosterically to control gene expression (Martinez-Zapien, Delsuc et al. 2014, Belorusova, Osz et al. 2016). The assembly of virus particles is of fundamental interest and potentially a process that can be targeted to disrupt the spread of disease causing viral pathogens. Margherita Marchetti (Vrije Universiteit Amsterdam, the Netherlands) presents an elegant single-molecule study where a combination of optical trapping and confocal microscopy (Hashemi Shabestari, Meijering et al. 2017) was used to visualize the self-assembly of artificial virus-like particles.  Belorusova, A. Y., J. Osz, M. V. Petoukhov, C. Peluso-Iltis, B. Kieffer, D. I. Svergun and N. Rochel (2016). "Solution Behavior of the Intrinsically Disordered N-Terminal Domain of Retinoid X Receptor alpha in the Context of the Full-Length Protein." Biochemistry 55(12): 1741-1748. Boehm, E. M., S. Subramanyam, M. Ghoneim, M. T. Washington and M. Spies (2016). "Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy." Methods Enzymol 581: 105-145. Cornish, P. V. and T. Ha (2007). "A survey of single-molecule techniques in chemical biology." ACS Chem Biol 2(1): 53-61. Daldrop, P., H. Brutzer, A. Huhle, D. J. Kauert and R. Seidel (2015). "Extending the range for force calibration in magnetic tweezers." Biophys J 108(10): 2550-2561. Duss, O., M. Yulikov, F. H. Allain and G. Jeschke (2015). "Combining NMR and EPR to Determine Structures of Large RNAs and Protein-RNA Complexes in Solution." Methods Enzymol 558: 279-331. Duss, O., M. Yulikov, G. Jeschke and F. H. Allain (2014). "EPR-aided approach for solution structure determination of large RNAs or protein-RNA complexes." Nat Commun 5: 3669. Fuentes-Perez, M. E., M. S. Dillingham and F. Moreno-Herrero (2013). "AFM volumetric methods for the characterization of proteins and nucleic acids." Methods 60(2): 113-121. Hamatsu, J., D. O'Donovan, T. Tanaka, T. Shirai, Y. Hourai, T. Mikawa, T. Ikeya, M. Mishima, W. Boucher, B. O. Smith, E. D. Laue, M. Shirakawa and Y. Ito (2013). "High-resolution heteronuclear multidimensional NMR of proteins in living insect cells using a baculovirus protein expression system." J Am Chem Soc 135(5): 1688-1691. Hashemi Shabestari, M., A. E. Meijering, W. H. Roos, G. J. Wuite and E. J. Peterman (2017). "Recent Advances in Biological Single-Molecule Applications of Optical Tweezers and Fluorescence Microscopy." Methods Enzymol 582: 85-119. Ito, Y., T. Mikawa and B. O. Smith (2012). "In-cell NMR of intrinsically disordered proteins in prokaryotic cells." Methods Mol Biol 895: 19-31. Lin, J. L., C. C. Wu, W. Z. Yang and H. S. Yuan (2016). "Crystal structure of endonuclease G in complex with DNA reveals how it nonspecifically degrades DNA as a homodimer." Nucleic Acids Res 44(21): 10480-10490. Martinez-Zapien, D., M. A. Delsuc, G. Trave, R. Lutzing, C. Rochette-Egly and B. Kieffer (2014). "Production and characterization of a retinoic acid receptor RARgamma construction encompassing the DNA binding domain and the disordered N-terminal proline rich domain." Protein Expr Purif 95: 113-120. Rutkauskas, M., A. Krivoy, M. D. Szczelkun, C. Rouillon and R. Seidel (2017). "Single-Molecule Insight Into Target Recognition by CRISPR-Cas Complexes." Methods Enzymol 582: 239-273. Sakakibara, D., A. Sasaki, T. Ikeya, J. Hamatsu, T. Hanashima, M. Mishima, M. Yoshimasu, N. Hayashi, T. Mikawa, M. Walchli, B. O. Smith, M. Shirakawa, P. Guntert and Y. Ito (2009). "Protein structure determination in living cells by in-cell NMR spectroscopy." Nature 458(7234): 102-105. Spies, M. (2014). "Fulfilling the dream of a perfect genome editing tool." Proc Natl Acad Sci U S A 111(28): 10029-10030. Szczelkun, M. D., M. S. Tikhomirova, T. Sinkunas, G. Gasiunas, T. Karvelis, P. Pschera, V. Siksnys and R. Seidel (2014). "Direct observation of R-loop formation by single RNA-guided Cas9 and Cascade effector complexes." Proc Natl Acad Sci U S A 111(27): 9798-9803. Taylor, J. A., C. L. Pastrana, A. Butterer, C. Pernstich, E. J. Gwynn, F. Sobott, F. Moreno-Herrero and M. S. Dillingham (2015). "Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation." Nucleic Acids Res 43(2): 719-731. Zhou, Q., H. Li, H. Li, A. Nakagawa, J. L. Lin, E. S. Lee, B. L. Harry, R. R. Skeen-Gaar, Y. Suehiro, D. William, S. Mitani, H. S. Yuan, B. H. Kang and D. Xue (2016). "Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization." Science 353(6297): 394-399.  

Protein-nucleic acids interactions: new ways of connecting structure, dynamics and function

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Maria Spies

Brian O. Smith

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Protein-nucleic acids interactions: new ways of connecting structure, dynamics and function

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