TY - JOUR
T1 - Fluorescence correlation spectroscopy of the binding of nucleotide excision repair protein XPC-hHr23B with DNA substrates
AU - Roche, Y.
AU - Zhang, D.
AU - Segers-Nolten, G. M.J.
AU - Vermeulen, W.
AU - Wyman, C.
AU - Sugasawa, K.
AU - Hoeijmakers, J.
AU - Otto, C.
N1 - Funding Information:
Acknowledgement This work was supported by the Dutch Technology Foundation (STW) through grant TTN.4821 and the European Community through a Human Frontiers in Science Program grant RGP0007/2004-C.
PY - 2008/9
Y1 - 2008/9
N2 - 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 (τ d) than for free DNA, indicating the presence of DNA-protein bound complexes. The fraction of DNA bound (θ), 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.
AB - 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 (τ d) than for free DNA, indicating the presence of DNA-protein bound complexes. The fraction of DNA bound (θ), 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.
KW - Fluorescence correlation spectroscopy
KW - Macromolecules interaction
KW - Nucleotide excision repair
KW - Oligonucleotides
UR - http://www.scopus.com/inward/record.url?scp=51649088122&partnerID=8YFLogxK
U2 - 10.1007/s10895-008-0390-6
DO - 10.1007/s10895-008-0390-6
M3 - Article
C2 - 18574675
AN - SCOPUS:51649088122
SN - 1053-0509
VL - 18
SP - 987
EP - 995
JO - Journal of Fluorescence
JF - Journal of Fluorescence
IS - 5
ER -