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

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.

We have carried out extensive kinetic characterization at ensemble level of the activity of archaeal 5' and 3' structure-specific endonucleases FEN1 and XPF in terms of substrate preference and we have already demonstrated an interaction between XPF and the sliding clamp PCNA, which hugely increases XPF activity while only stimulating FEN1 by 50-fold. To further advance in our understanding of substrate recognition and unravel the sequence of events and conformational changes involved in the formation of the active complex, we wish to apply single-molecule fluorescence techniques. Our main objectives are: 1) Characterise the nuclease-induced distortion for different DNA substrates (flaps, nicked duplexes, replication forks, nicked 4-way junctions). 2) Quantify the affinity, the lifetime of the complex nuclease-substrate and characterise the large domain rearrangement observed in the crystal structures of XPF and FEN1 bound to duplex DNA. 3) Determine the orientation of the DNA in the complex substrate-nuclease and compare it with the models derived from molecular dynamics and crystallographic data. 4) Investigate the mode of interaction between each enzyme and PCNA in the absence and presence of the substrate and look for evidences about PCNA-induced conformational changes in the complex nuclease-substrate that may lead to nuclease activation. 5) Use single-molecule enzymology to quantify the effect that specific mutations have on the individual rate constants. We have demonstrable expertise in the biochemical and single-molecule techniques required and we have cloned and purified XPF, FEN1 and PCNA, constructed dye-labelled DNA substrates, and already optimised binding and cleavage ensemble FRET assays. In summary, this is an ambitious proposal with clear, achievable aims. We expect to deliver important new insights to fundamental DNA processing pathways and generate significant research findings that will be published in high impact journals.