Fluorescence correlation spectroscopy of the binding of nucleotide excision repair protein XPC-hHr23B with DNA substrates.
The interaction of the nucleotide excision repair (NER) protein dimeric complex XPC-hHR23B, which is implicated in the DNA damage recognition step, with three Cy3.5 labeled 90-bp double-stranded DNA substrates (unmodified, with a central unpaired region, and cholesterol modified) and a 90-mer single-strand DNA was investigated in solution by fluorescence correlation spectroscopy. Autocorrelation functions obtained in the presence of an excess of protein show larger diffusion times (tau (d)) than for free DNA, indicating the presence of DNA-protein bound complexes. The fraction of DNA bound (theta), as a way to describe the percentage of protein bound to DNA, was directly estimated from FCS data. A significantly stronger binding capability for the cholesterol modified substrate (78% DNA bound) than for other double-stranded DNA substrates was observed, while the lowest affinity was found for the single-stranded DNA (27%). This is in accordance with a damage recognition role of the XPC protein. The similar affinity of XPC for undamaged and 'bubble' DNA substrates (58% and 55%, respectively) indicates that XPC does not specifically bind to this type of DNA substrate comprising a large (30-nt) central unpaired region.
Roche
Y
Y
Biophysical Engineering Group, Faculty of Science and Technology, MESA+ Institute for Nanotechnology and BMTI, University of Twente, Enschede, The Netherlands.
Zhang
D
D
Segers-Nolten
G M J
GM
Vermeulen
W
W
Wyman
C
C
Sugasawa
K
K
Hoeijmakers
J
J
Otto
C
C
D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C513287:RAD23B protein, human; C086984:XPC protein, human; D004247:DNA; D045643:DNA Repair Enzymes