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New light on the compaction of DNA in cells (article in Cell Reports by Rifka Vlijm, Cees Dekker, and Nynke Dekker (BN) and others)

[12-01-2015]

(by: TU Delft/Communication TNW)
Researchers of the Kavli Institute of Nanoscience of TU Delft have discovered that tetrasomes - proteins that play a crucial role in the folding of DNA - fluctuate spontaneously between a left-handed and a right-handed form. This discovery has changed our view of the organization of DNA in cells. The research, conducted in cooperation with the Innsbruck Medical University, has appeared this week in the journal Cell Reports.

Nucleosomes

The length of our DNA (about 1 meter) is enormous compared to the dimensions of a cell nucleus (several micrometers). To ensure that DNA fits in the cell, it is systematically wrapped around certain proteins, resulting in the formation of nucleosomes. However, this conglomeration of DNA by nucleosomes also decreases the accessibility of the DNA for reading out the genes, for example. A dynamic interplay between DNA and proteins leads to a good balance between compact DNA and accessible DNA, but this interplay is still not properly understood.

 

Figure 1: Raw data, with illustrations of the effects. The data at the bottom shows the observed rotations of the ball (Lk = linking number expressed as the number of rotations) as a function of time (in seconds). The experiment starts with a bare piece of DNA. A tetrasome is formed after the addition of half of the histones. The rotation of the magnetic ball changes when the DNA is wrapped around the proteins. The ball keeps rotating back and forth, which means that the form of the tetrasome is constantly changing. This continues until the researchers add the missing proteins. A stable nucleosome is then formed once the DNA is wrapped a little further around the protein disk.

Magnetic tweezers

The Delft researchers can now study the dynamics of a single nucleosome on DNA. They succeeded in forming artificial nucleosomes on an individual piece of DNA while simultaneously measuring different properties of the DNA. 
The method used is a recent variation of a magnetic tweezers technique, in which a single DNA molecule is attached to a glass plate at one end and to a magnetic ball at the other. The placement of a doughnut-shaped magnet directly above the magnetic ball not only extends the DNA, but also enables the magnetic ball to rotate freely, allowing spontaneous rotations of the DNA to be read out. 
By taking extremely accurate measurements of both the height and the rotation of the magnetic ball before, during and after the formation of nucleosomes and tetrasomes (intermediate forms of nucleosomes), the researchers succeeded in determining the stability of these structures. 

Figure 2: The significance of an observed rotation of the magnetic ball. Only the magnetic ball can be observed with the camera; DNA is too small to be seen. This figure illustrates the significance of the movement of the magnetic ball: The tetramer changes from left-handed to right-handed, while the length of the DNA does not increase. The length of the DNA (blue) remains constant. 

Waving arms

While taking measurements, the researchers discovered that a nucleosome is formed in two steps. In the first step, a tetrasome is formed from four so-called histone proteins, while the second step involves the addition of four histones to create a complete nucleosome containing 50 nanometres of DNA wrapped around the protein disk in a leftward direction.
As expected, the nucleosome was very stable and even remained that way for hours. By only adding half of the proteins, however, it became possible to study the tetrasome intermediate form for the first time. This yielded a surprise - the rotation continued back and forth, even though the length of the DNA did not change any more. The rotation direction of the tetrasome kept spontaneously changing. A model of structural change within the tetrasome, the so-called 'waving arms model', has been created to explain this movement.

 

Figure 3: The so-called 'waving arms model'. Top: the spontaneous change in structure of a tetrasome. Bottom: the movement made by the arms of a car driver when the wheels are turned from right to left. This figure illustrates the similarity between the structure of the tetramer protein and the arms of the driver. 

Reading out

The fact that reading out the genes in the DNA causes a great deal of twist in the DNA is a problem in cell biology that has remained unsolved for a long time. This applied twist causes problems for the functioning of the motor proteins that need to do their work on the DNA. Nevertheless, this does not seem to cause any difficulties for living cells. The fact that tetrasomes are able to 'absorb' this twist now seems to offer a good explanation for this robust functioning of our DNA.

This study was led by doctoral candidate Rifka Vlijm and post-doc Mina Lee and involved collaboration between the TU Delft groups of prof. Cees Dekker, who initiated the nucleosome research, and prof. Nynke Dekker, who developed the special magnetic tweezers measurement technique. The proteins were purified by prof. Alexandra Lusser of Innsbruck Medical University. 

Figure 4: Research summary. Bottom: the discovery of the structural change of a tetrasome. Top: the change that actually occurs during the experiment. The background shows the most common form of a tetrasome. 

Further reading
Rifka Vlijm*, Mina Lee*, Jan Lipfert, Alexandra Lusser, Cees Dekker, and Nynke H. Dekker (*=equal contribution). “Nucleosome assembly dynamics involve spontaneous fluctuations in the handedness of tetrasomes”  Cell Reports 10, 1-10, January 13, 2015

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