Ultraviolet light, tobacco smoke or even the benzopyrenes contained in over-cooked meat can cause changes to the DNA in our cells, which may lead to the onset of cancers. These environmental agents deteriorate the actual structure of the DNA, notably causing so-called "bulky" lesions (like the formation of chemical bonds between DNA bases). In order to identify and repair this type of damage, the cell can call on several systems, such as transcription-coupled repair (TCR), whose complex mechanism of action still remains poorly understood today. Abnormalities affecting this TCR mechanism – which permits permanent monitoring of the genome – are the cause of some hereditary diseases such as Xeroderma pigmentosum
, sufferers from which are hypersensitive to the Sun's ultraviolet rays and are commonly referred to as "children of the night".
For the first time, a team from Institut Jacques Monod (CNRS/Université Paris Diderot), in collaboration with scientists at the Universities of Bristol in the UK and Rockefeller in the USA, has succeeded in observing the initial stages of TCR repair mechanisms in a bacterial model. To achieve this, they employed a novel technique for the nanomanipulation of individual molecules(1) which allowed them to detect and follow real-time the interactions between the molecules in play in a single damaged DNA molecule. They elucidated the interactions between different actors during the first steps of this TCR process. A first protein, RNA polymerase(2), usually crosses DNA without mishap, but is stalled when it meets a bulky lesion (like a train blocked on its rails by a landslide). A second protein, Mfd, binds to the stalled RNA polymerase and removes it from the damaged "rail" so that it can then replace it with the other proteins necessary to repair the damage. Measurements of the reaction speeds enabled the observation that Mfd acts particularly slowly on RNA polymerase, pushing it out of the way in about twenty seconds. Furthermore, Mfd does indeed displace stalled RNA polymerase, but then remains associated with the DNA for a longer period (of about five minutes), allowing it to coordinate the arrival of other repair proteins at the damaged site.
Although the scientists were able to explain how this system can achieve almost 100% reliability, a even clearer understanding of these repair processes is still essential in order to determine how cancers appear and subsequently may become resistant to chemotherapies.
(1) During these nanomanipulation experiments, damaged DNA was grafted onto a glass surface on one side and a magnetic microbead on the other. The bead surface enabled the perpendicular extension of the DNA and measurement of this end-to-end extension using videomicroscopy. The binding to DNA of different proteins, and their action, is identifiable from the modification the protein generates in the structure or conformation of the DNA. This technique enables an extremely detailed structural and kinetic analysis of in vitro biochemical reactions.
(2) RNA polymerase is responsible for the reading of DNA by a gene and its rewriting in an RNA form, a process know as transcription. It has been shown that RNA polymerase does not only transcribe genes, but also the DNA between genes (until recently referred to as "junk" DNA), allowing, for example, polymerase RNA to perform its quality control by TCR on the entire genome of an organism.
“Initiation of transcription-coupled repair characterized at single-molecule resolution”
Kevin Howan, Abigail J. Smith, Lars F. Westblade, Nicolas Joly, Wilfried Grange, Sylvain Zorman, Seth A. Darst, Nigel J. Savery et Terence R. Strick
Nature, 9 September 2012