The genetic material of most organisms is carried by DNA, a complex organic molecule. DNA is very long — for humans, the molecule is estimated to be about 2 m in length. In cells, DNA occurs in a densely packed form, with strands of the molecule coiled up in a complicated but efficient space-filling way. A key role in DNA’s compactification is played by histones, structural-support proteins around which a part of a DNA molecule can wrap. The DNA–histone wrapping process is reversible — the two molecules can unwrap and rewrap — but little is known about the mechanisms at play. Now, by applying high-speed atomic-force microscopy (HS-AFM), Richard Wong and colleagues from Kanazawa University (NanoLSI WPI) provide valuable insights into the spatiotemporal dynamics of DNA–histone interactions.
The researchers looked at the interaction between DNA and a histone called H2A, one of the five main histones. To check the applicability of HS-AFM as a viable tool for imaging the DNA–histone interaction, they first focused on H2A in its native state. Wong and colleagues were able to image the topology of the molecule, and how it changes over time. Importantly, they showed that the HS-AFM process, during which a tapping force is constantly exerted on the molecule, does not lead to conformational changes or actual damage.
For real-time observation of the DNA–H2A interaction with HS-AFM, the scientists prepared DNA samples with different lengths and forms: plasmid (long and circular), long-linearized and short-linearized DNA, with the latter having the highest motility. The experiments showed that the choice of substrate on which to put the DNA for AFM imaging is crucial; a particular type of lipid layer was found to be good as it does not strongly absorb DNA strands.
The observations of the interaction of H2A with short-linearized DNA, which the researchers nicknamed ‘inchworm DNA’, led to the most notable results. Specifically, four different interaction situations could be distinguished: touching, sliding, sandwiching and wrapping, with the associated motions indeed resembling the movements of inchworms.
Wong and colleagues also investigated the effect of ionic strength on the DNA–histone binding affinity, by changing the salt concentration of the liquid containing the DNA–histone aggregate. When increasing the liquid’s salinity, the aggregate was found to dissolve. When diluting the liquid again — and so reducing the salt content — the aggregate reformed. This result shows that varying the ionic strength (i.e., the salt concentration) of the environment of the DNA–H2A complex provides a way to mimic the variations in the strength of DNA–histone interactions as they happen in living organisms.
The report of Wong and colleagues represents the first real-time observation of DNA–histone interactions, and convincingly shows the applicability of HS-AFM for studying this kind of biological process, also in the context of diseases. Quoting the researchers: “[Our work] demonstrates … the potential to study protein aggregation and protein–nucleic acid aggregate formation in various human diseases.”
Finally, it is worth highlighting the contribution of the paper’s first author, Goro Nishide, who is a pre-doctoral student at the Division of Nano Life Science in the Graduate School of Frontier Science Initiative at Kanazawa University. Mr Nishide played a key role in the reported research, supervised by Professor Wong and Dr. Lim, by performing the experiments, co-designing the study and co-writing the paper. Mr Nishide is also enrolled in Kanazawa University’s WISE program for Nano-Precision Medicine, Science and Technology, an initiative aimed at innovations in disease prevention, diagnosis, and treatment methods based on exploiting our increased understanding of biological and other processes at the nanoscale.
Real-time visualization of DNA wrapping around histone H2A using HS-AFM A DNA strand initially slides on histone H2A, then sequentially adopts Sandwiching and Wrapping topologies (scale bar: 30nm).
Direct real-time visualization of histone H2A-DNA complex aggregation, dissolution, and re-aggregation using HS-AFM. Long period observation of a histone H2A short-linearized dsDNA complex. Initially, histone H2A and short-linearized dsDNA were constantly added into scanning buffer to promote formation of large aggregate on bare mica. Then, ionic strength of scanning buffer was increased by changing scanning buffer containing different concentrations of salt. Dissolution of the aggregate was indicated with a white asterisk. The change was reversible upon replacing with salt-free buffer (scale bar: 75nm).
Dynamic inchworm-wrapping interactions between short-linearized dsDNA and histone H2A on a cationic lipid substrate captured using HS-AFM.
Atomic force microscopy
Atomic force microscopy (AFM) is an imaging technique in which the image is formed by scanning a surface with a very small tip. Horizontal scanning motion of the tip is controlled by piezoelectric elements, while vertical motion is converted into a height profile, resulting in a height distribution of the sample’s surface. As the technique does not involve lenses, its resolution is not restricted by the so-called diffraction limit as in X-ray diffraction, for example. In a high-speed setup (HS-AFM), the method can be used to produce movies of a sample’s structural evolution in real time, as a typical biomolecule can be scanned in 100 ms or less. Now, Richard Wong and colleagues from Kanazawa University have successfully applied the HS-AFM technique to study the wrapping of DNA around structural proteins.
Division of Nano Life Science in the GRAFINITI and WISE program at Kanazawa University
Goro Nishide, the first author of the paper reporting the application of HS-AFM for visualizing DNA–histone dynamics, is a pre-doctoral student at the Division of Nano Life Science in the Graduate School of Frontier Science Initiative (GRAFINITI), an educational program at Kanazawa University fostering excellent graduate students wishing to participate in establishing the new scientific field of “Nano Probe Life Science”. The students’ supervisors for this program are all world-class researchers affiliated to the WPI-Nano Life Science Institute (WPI-NanoLSI), a research center established in 2017 as part of Japan’s World Premier International Research Center Initiative (WPI) of the Ministry of Education, Culture, Sports, Science and Technology, the objective of which is the creation of world-tier research centers.
In addition, Mr Nishide is enrolled in Kanazawa University’s WISE Program for Nano-Precision Medicine, Science, and Technology. Notably, WISE is a five-year integrated pre- and post-doctoral degree program that focuses on five types of diseases in humans (cancer, lifestyle diseases, neurological diseases, diseases caused by small particulates, and diseases caused by nanomaterials). Its mission is to foster technically skilled medical and engineering professionals who all contribute to the creation of innovative disease prevention, diagnosis, and treatment methods through understanding and control on the nanoscale level. The WISE program is nurtured by a community of scientists from four domains (nanometrology, life sciences, supramolecular chemistry, and computational science), all engaging in transdisciplinary dialogue.
This work was supported by grants from MEXT/JSPS KAKENHI Grant: 19K23841, 20K16262, 17H05874 and 17K08655, the Kobayashi International Scholarship Foundation, and the Shimadzu Science Foundation.
Title: High-Speed Atomic Force Microscopy Reveals Spatiotemporal Dynamics of Histone Protein H2A Involution
Journal:The Journal of Physical Chemistry Letters