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3D Structure of Biologically Active DNA Revealed in Unprecedented Detail; Myriad Beautiful & Unexpected Shapes Seen in Dynamic Super-Coiled DNA; Hinges May Make Tightly Wound DNA More Open Than Thought

Researchers at the Baylor College of Medicine (USA) and the University of Leeds (UK) have imaged, in unprecedented detail, the three-dimensional structure of supercoiled DNA, revealing that its shape is much more dynamic than the well-known linear double-helix. Various DNA shapes, including figure-8's, were imaged using a powerful microscopy technique by researchers at Baylor, and then examined using supercomputer simulations run at the University of Leeds. As reported online on October 12, 2015 in an open-access article published in Nature Communications, the simulations also show the dynamic nature of DNA, which constantly wiggles and morphs into different shapes - a far cry from the commonly held idea of a rigid and static double-helix structure. Improving the understanding of what DNA looks like when it is in the cell will help us to design better medicines, such as new antibiotics or more effective cancer chemotherapies, the researchers suggest. "This is because the action of drug molecules relies on them recognizing a specific molecular shape - much like a key fits a particular lock," said Dr. Sarah Harris from the School of Physics and Astronomy at the University of Leeds, who led the computer simulation research side of the study. The Nature Communications article is titled “'The Structural Diversity of Supercoiled DNA.” The double-helix shape has a firm place in the public's collective consciousness. It is referenced in popular culture and often features in art and design. But the shape of DNA isn't always that simple. Dr. Harris said: "When Watson and Crick described the DNA double-helix, they were looking at a tiny part of a real genome, only about one turn of the double helix. This is about 12 DNA base pairs, which are the building blocks of DNA that form the rungs of the helical ladder. Our study looks at DNA on a somewhat grander scale - several hundreds of base pairs - and even this relatively modest increase in size reveals a whole new richness in the behavior of the DNA molecule."

There are actually about 3 billion base pairs that make up the complete set of DNA instructions in humans. This is about a meter of DNA. This enormous string of molecular information has to be precisely organized by coiling it up tightly so that it can be squeezed into the nucleus of cells.

To study the structure of DNA when it is packed into cells, the researchers needed to replicate this coiling of DNA.

"The beautiful double-helical structure we all know and love is not the actual active form of DNA," said Dr. Lynn Zechiedrich, Professor in the Department of Molecular Virology and Microbiology at Baylor, and co-contributing author with Dr. Wah Chiu, Professor in the Verna and Marrs McLean Department of Biochemistry and Molecular Biology, also at Baylor.

"Previous studies were on short fragments (6-12 base pairs) of linear DNA, but human DNA is constantly moving around in your body - and it coils and uncoils,” said Dr. Zechiedrich. “You can't coil linear DNA and study it, so we had to make circles so the ends would trap the different degrees of winding."

Dr. Chiu and Dr. Zechiedrich, collaborating with Dr. Steven Ludtke and Dr. Michael Schmid, also of Baylor, and Dr. Harris, examined tiny DNA mini-circles (now known as "MiniVectors," a named coined by the team and commercially available) containing only 336 base pairs, using methods from chemistry, physics, math, and computer modeling.

To investigate how the winding affected what the circles looked like, the researchers wound and then unwound the tiny DNA circles, 10 million times shorter in length than the DNA contained within our cells, a single turn at a time.

Each cell in the human body holds about a meter of DNA (ten million times longer than the tiny circles the team made). In their study, the researchers investigated how the winding process affected what the circles looked like, using very powerful microscopes.

The researchers also devised a test to make sure that the tiny twisted-up DNA circles that they made in the lab were biologically active. They used purified human topoisomerase II alpha, an essential enzyme that manipulates DNA and important target of anticancer drugs.

This enzyme relieved the winding stress from all of the supercoiled minicircles, even the most coiled ones, which is its normal job in the human body. This result means that the circles must look and act like the much longer DNA that topoisomerases encounter in human cells.

"These enzymes don't do anything to linear DNA because it's not coiled up," said co-author Dr. Daniel J. Catanese, Jr., also of Baylor.

Dr. Rossitza Irobalieva, the co-lead author on the publication, who conducted the work while she was at Baylor, used “cryo-electron tomography” - a powerful microscopy technique that involves first freezing biologically active material - to provide the first three-dimensional images of individual circular DNA molecules.

She saw that coiling the tiny DNA circles caused them to form a variety of beautiful and unexpected shapes.

"Some of the circles had sharp bends, some were figure-8’s, and others looked like handcuffs or racquets or even sewing needles. Some looked like rods because they were so coiled," said Dr. Irobalieva.

The static images produced by the cryo-electron tomography were then compared to, and matched with, shapes generated in supercomputer simulations that were run at the University of Leeds.

These simulated images provided a higher-resolution view of the DNA and showed how its dynamic motion makes its shape constantly change to form a myriad of structures.

The cryo-electron tomography of the tiny DNA circles also revealed another surprising finding.

Base pairs in DNA are like a genetic alphabet, in which the letters on one side of the DNA double-helix only pair with a particular letter on the other side.

While the researchers expected to see the opening of base pairs - that is, the separation of the paired letters in the genetic alphabet - when the DNA was under-wound, they were surprised to see this opening for the over-wound DNA. They were surprised because over-winding is supposed to make the DNA double-helix stronger, and theoretically less likely to open.

The researchers hypothesize that this disruption of base pairs may create flexible hinges, allowing the DNA to bend sharply, perhaps helping to explain how a meter of DNA can be packed into a single human cell.

Dr. Harris concludes, "We are sure that supercomputers will play an increasingly important role in drug design. We are trying to do a puzzle with millions of pieces, and they all keep changing shape."

"Being able to observe individual DNA circles allows us to understand the different structures of biologically active DNA. Each of these different structures facilitates how DNA interacts with proteins, other DNA and RNA, and anti-cancer drugs, adapting to the cell processes required," said Dr. Jonathan Fogg, the other lead author of the publication, also of the Baylor College of Medicine.

"The next step is to start adding the other components of the cell or anticancer drugs to see how the DNA shapes change," said Dr. Fogg.

Additional contributors to the study include Muyuan Chen, Anna K. Barker, of Baylor College of Medicine, and Dr. Thana Sutthibutpong, of the University of Leeds.

The MiniVectors were made available by Twister Biotech, which now produces them commercially.

IMAGES

The image shows the structure of the DNA calculated with the supercomputer simulations (in color) superimposed upon the cryo-electron tomography data (in white or yellow). (There is no superimposition onto cryo-electron tomography data for the purple figure-8 shape.) You can see that the familiar double-helix has been either simply bent into a circle or twisted into a figure-8. (Credit: Dr.Thana Sutthibutpong).

VIDEOS

A video (see link below) illustrates how supercomputer simulations show how the dynamic motion of the supercoiled DNA causes its shape to change constantly to form a myriad of structures. (Credit: Dr. Thana Sutthibutpong).

An entertaining video from the Zechiedrich lab on DNA base pairs is also provided (see link below).

[Baylor press release] [Leeds press releasel] [Nature Communications article] [Video of supercomputer simulations ] Entertaining YouTube video from Zechiedrich lab] [Twister Biotech]