DNA damage often occurs as a by-product of normal cellular metabolic functions. Fortunately, cells have evolved multiple mechanisms to remove damaged DNA extremely efficiently as aberrant repair pathways have been strongly linked to various disease states, especially cancer. One specific DNA damage that occurs most often is when the phosphate bond on DNA strand, which links two adjacent nucleotide bases, becomes broken—a process scientists refer to as single stand break (SSBs).
Now, a collaborative team of researchers has discovered a new mechanism cells employ in order to correct SSBs during transcription through chromatin. Understanding the data from this study is critical, since unrepaired SSBs are a major threat to genomic stability and can drastically interfere with DNA transcription, replication, and repair.
“In higher organisms DNA is bound with proteins in complexes called the nucleosome. Every approximately 200 base pairs are organized in nucleosomes, consisting of eight histone proteins, which, like the thread on the bobbin, wound double helix of DNA, which is coiled into two supercoiled loops. Part of the surface of the DNA helix is hidden because it interacts with histones. Our entire genome is packed this way, except for the areas, from which the information is being currently read”, explained senior author Vasily Studitsky, Ph.D., head of the Laboratory of Regulation of Transcription and Replication at Lomonosov Moscow State University.
The findings from this study were published recently in Science Advances through an article entitled “Structure of transcribed chromatin is a sensor of DNA damage.”
The dense packing of DNA allows the cell to fit the entire molecule, which is serval times longer than the length of the cell, within its membranous constraints. However, the coiling and packaging process makes it extremely difficult for DNA repair mechanisms to gain access to the damaged strands. The investigators were interested in how cells manage to repair this sequestered DNA, specifically during transcription.
The investigators observed in vitro, using an RNA polymerase enzyme along with DNA packaged into nucleosomes, that the polymerase sensed the SSB just after it had transcribed over the break, stopping its forward progression—a signal typically picked up by the cell which signals DNA damage response elements to be recruited to the site of the polymerase stall.
“We have shown, not yet in the cell, but in vitro, that the repair of breaks in the other DNA chain, which is “hidden” in the nucleosome, is still possible. According to our hypothesis, it occurs due to the formation of special small DNA loops in the nucleosome, although normally DNA wounds around the histone “spool” very tightly,” stated Dr. Studitsky. “The loops form when the DNA is coiled back on nucleosome together with the polymerase. RNA polymerase can “crawl” along the DNA loops nearly as well as on histone-free DNA regions, but when it stops near locations of the DNA breaks, it “panics,” triggering the cascade of reactions to start DNA “repairs.”
Analysis of DNA breaks at different positions on the in vitro strands allowed the researchers to hypothesize that the stalling of RNA polymerase was caused by the formation of the loop, which blocks movement of the enzyme—a mechanism previously uncharacterized for nucleosome DNA.
“In terms of applied science, the discovery of a new mechanism of reparation promises new prospective methods of prevention and treatment of diseases. We have shown that the formation of loops, which stop the polymerase, depends on its contacts with histones. If you make them more robust, it will increase the efficiency of the formation of loops and the probability of repair, which in turn will reduce the risk of disease. If these contacts are destabilized, then by using special methods of drug delivery you can program the death of the affected cells,” Dr. Studitsky concluded.