Nucleic Acids Research, 2002, Vol. 30, No. 21 4720-4727
© 2002 Oxford University Press
Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes
Department of Applied Physics, Biophysical Technology Group, Biomedical Technology Institute, University of Twente, PO Box 217, 7500 AE Enschede, The Netherlands, 1 Department of Cell Biology and Genetics, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands and 2 Department of Radiation Oncology, University Hospital Rotterdam-Daniel, Rotterdam, The Netherlands
*To whom correspondence should be addressed. Tel: +31 53 489 3358; Fax: +31 53 489 1105; Email: g.m.j.segers-nolten{at}tn.utwente.nl
We used scanning confocal fluorescence microscopy to observe and analyze individual DNA– protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measure ments were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level 
10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.