Repair of "hidden" breaks in the DNA chain
A loop for all diseases
The mechanism of DNA repair discovered by Russian scientists
it will help to treat and prevent diseasesEkaterina Mishchenko, Vladimir Koryagin, "Gazeta.
The DNA molecule in human cells is chemically unstable, which causes its damage of various nature. There are a number of mechanisms that provide a response to DNA damage, which includes the processes of detecting DNA damage, generating a damage signal and "repairing" – the so–called repair - of the DNA molecule.
Previously, "Gazeta.Ru" told about the fact that the prestigious scientific journal PNAS published an article by Svetlana Khoronenkova, a doctoral student of the Faculty of Chemistry at Moscow State University, devoted to how DNA "repairs" itself and how DNA breaks are related to genetic diseases ("Signaling for damaged DNA"). Now, a group of researchers from Lomonosov Moscow State University, led by Professor Vasily Studitsky, together with their foreign colleagues, have discovered a new mechanism for repairing DNA. This mechanism opens up new prospects for the treatment and prevention of neurodegenerative diseases, for example, Alzheimer's disease, which is still incurable.
An article describing the discovery was published in the scientific journal Science Advances, founded by the publisher of the journal Science in February 2015 (Pestov et al., Structure of transcribed chromatin is a sensor of DNA damage).
"In higher organisms, DNA is bound into complexes with proteins – nucleosomes. For every 200 pairs of nucleotides, there is a DNA-protein complex – a nucleosome consisting of 8 histone proteins - on which, like a thread on a coil, a double helix of DNA is wound, folded into two superspiral turns. Part of the surface of the DNA helix is hidden because it interacts with histones. This is how our entire genome is packed, except for the areas from which information is currently being read," says Vasily Studitskiy, Doctor of Biological Sciences, a leading researcher and head of the Laboratory for Transcription Regulation and Replication of the Faculty of Biology of Moscow State University, who received a megagrant in 2010.
The dense packaging allows a thread about two meters long to fit into a microscopic cell nucleus, but makes significant DNA surfaces inaccessible to repair enzymes – proteins that trigger the mechanisms of "repairing" damaged areas. And DNA damage leads to the accumulation of mutations, cell death, and at the level of the whole organism to various diseases, including neurodegenerative, such as Alzheimer's disease.
The group, headed by Professor Vasily Studitskiy, studied the mechanism of recognition by enzymes of single-stranded DNA breaks, in which the connection between the nucleotides of one of the chains is lost, in places where DNA is bound to histones.
Scientists know quite a lot about reparations at active sites. As is known, for protein synthesis, the genetic code – instructions for its assembly, where triplets of nucleotides correspond to a certain amino acid – must be carried out from the nucleus into the cytoplasm of the cell.
A thin and long strand of DNA is packed in the nucleus and can break when it comes out, but it cannot be sacrificed: nuclear DNA is in a single copy in the cell.
Therefore, when it is necessary to synthesize a certain protein, its small section unwinds, two chains open and information about the structure of the protein from one of the DNA chains is rewritten to RNA, a small single-stranded molecule. A molecule of matrix (informational) RNA, which becomes an indication of how to make a protein, is synthesized according to the principle of complementarity: each nucleotide corresponds to a paired one.
During transcription (reading) of information and rewriting it to RNA, the RNA polymerase enzyme "rides" along the DNA chain and, having detected a break, stops. As a text corrector, RNA polymerase, having stopped rewriting, triggers a cascade of reactions, as a result of which repair enzymes correct the damaged area. At the same time, RNA polymerase cannot detect breaks present in another DNA chain.
"We have proved (not yet in a cell, but in a test tube, in vitro) that the repair of breaks in another DNA chain, "hidden" in the nucleosome, is still possible. According to our assumption, this is due to the formation of special small DNA loops on the nucleosome, although normally the thread is wound very tightly on the histone "coil", – said Vasily Studitsky. – They occur when DNA is wound back onto the nucleosome along with polymerase. RNA polymerase can "crawl" along such DNA loops, as well as on a histone-free site, and, stopping near the places of DNA breaks, "raise panic", launching a cascade of reactions to start "repair work".
During the experiment, sections were introduced into the DNA that can be easily artificially broken by adding special enzymes to the test tube. After that, a system with a single nucleosome and transcription with a single RNA molecule was created. Thanks to the model system developed in 2002 by the same group of scientists, histones were planted on a molecule with an accuracy of one nucleotide. Having specially inserted breaks in precisely defined places on the DNA, the scientists investigated the effect of breaks on the speed of movement of RNA polymerase.
It turned out that it was in the nucleosomes, and not on histone-free DNA, that the enzyme stopped when a break was present in another DNA chain. Moreover, he stopped not before the break, but immediately after it. It was quite difficult to understand the mechanism that allows him to notice damage behind his "back", as if he had "eyes on the back of his head", although, obviously, he has neither.
The proposed structure of the DNA nucleosomal loop, which is temporarily formed during transcription of chromatin containing intact DNA by RNA polymerase 2. In the presence of a break in the DNA chain, the loop structure probably changes, preventing the rotation of the RNA polymerase along the DNA helix (orange arrow). Picture: Nadezhda GerasimovaAnalysis of breaks in various positions allowed us to hypothesize that the stopping of the movement of RNA polymerase causes the formation of a loop that blocks its path.
The data obtained open up a new direction for work on the topic of DNA repair.
Previously, the role of chromatin was considered passive: it was believed that only in the unfolded DNA strand "repair work" could occur. But Studitskiy and his colleagues found out that it is possible to repair the thread without unwinding the "coil" and highly conserved histones – changes in their structure are rejected by natural selection – play an important role in this.
And the high conservativeness of the protein just implies its active participation in many processes.
In addition, in the models proposed in the paper, scientists for the first time explain the role of the so-called topological locks, which are formed when any enzyme passes through DNA at the moment when it meets a nucleosome.
"From the point of view of applied science, the discovery of a new mechanism of reparation promises new promising methods of preventing and treating diseases. We have shown that the formation of loops that stop polymerase involves its contacts with histones. If you make them more durable, the efficiency of loop formation and the probability of repair will increase, which will reduce the risk of diseases. If these contacts are destabilized, then with the help of special methods of drug delivery, it is possible to program the death of affected cells," concluded Vasily Studitsky, adding that the process of developing and testing such drugs, of course, will take a lot of time.
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06.07.2015