Organization and function of structure-specific endonucleases: single-molecule studies of fluorescently labelled NER complexes

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Since a few years ago, tremendous technical developments in the detection of very low levels of light have made it possible to detect, track, and manipulate single biomolecules. The trick is to incorporate into the biomolecule a label, another molecule that emits fluorescence when excited by a light source like a laser. Although we can not see the biomolecule itself, we can identify its position by detecting the fluorescence signature coming from the label attached to it. Thus, we can differentiate molecules by labelling them with different fluorescence colours and use this property to investigate how molecules interact and for how long. Also, we can label different parts of the same molecule with different colours to get information about their relative movement. This has made single-molecule fluorescence a particularly powerful technique in elucidating mechanisms of molecular machineries: what they do, how they work individually, how they work together, and finally, how they work inside live cells. We want to apply this technique to study the DNA repair machinery. DNA repair is a very important task and cells devote a lot of energy to this, as mutated DNA or wrong DNA structures can cause severe damage in living organisms if they are copied and propagate. We have studied some of the proteins that participate in this repair mechanism, in particular structure-specific endonucleases such as XPF and FEN1 that recognize anomalous DNA structures and cut DNA strands protruding outside the double helix. These proteins are derived from archaeal organisms, a group of microbes that are very useful models because their DNA processing pathways are rather similar but simpler than those in higher organisms (yeast, worms and humans). We know the structure of these proteins and we have characterized them by conventional techniques, where you look at millions of copies at the same time. However, to further advance in our understanding of these mechanisms we need to extract the information that is only accessible by looking one molecule at a time, in which order they interact, for how long they remain attached to the damage DNA and how they recognize the anomalous DNA structures.