Single molecule fluorescence microscopy on Nucleotide Excision Repair complexes using GFP fusion proteins

Scanning Confocal Fluorescence Microscopy is used for single molecule studies on DNA-protein complexes that occur in Nucleotide Excision Repair (NER). During DNA-damage elimination by the NER-pathway, complex protein structures assemble over DNA. It is our aim to resolve the architecture of these DNA-protein complexes and to study dynamic changes that occur in these structures. For this purpose NER-complexes are partly reconstituted onto DNA-substrates using NER-proteins fused to different Green Fluorescent Protein (GFP) mutants. Here we describe the recombinant production of NER-GFP fusion proteins. Characterization of GFP fluorescence is shown together with results of GFP single molecule imaging. First results with NER-GFP fusion proteins are presented as well.