The XPC-HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay.
The XPC-HR23B complex is a prime candidate for the initial damage recognition step during global genome nucleotide excision repair. A specific interaction between the XPC-HR23B complex and various types of damaged DNA substrates has been demonstrated in recent work by electrophoretic mobility shift assays or immunoprecipitation. Although these studies allowed the estimation of relative binding affinities for the different types of lesions, the presence of large amounts of competitor DNA or the need for glutaraldehyde fixation prevented the quantification of equilibrium constants. We have performed a quantitative study on the binding of XPC to damaged DNA using fluorescence anisotropy measurements. The XPC-HR23B complex binds with high affinity (K(D) approximately 1-3 nM) to fluorescent 36 bp DNA fragments containing a single cisplatin 1,3-intrastrand adduct or a six-nucleotide mispaired region. From stoichiometric titration experiments, it is concluded that approximately 70% of the XPC-HR23B preparation is active in DNA binding. Binding experiments employing fluorescent probes with a single defined photoproduct reveal a 30-fold preference of XPC for 6,4-photoproducts as compared to a cyclobutane dimer. Competition experiments with undamaged and damaged plasmid DNA indicate that the XPC-HR23B complex discriminates between damaged and undamaged sites with high specificity. The specificity factor is between 100 and 3000, depending on the number of nonspecific sites considered in the calculations. Upon addition of XPA to the XPC binding reaction mixtures, it was not possible to detect cooperative ternary complex formation on the platinated 36 bp probe.
Hey
Thomas
T
Lehrstuhl für Biochemie, Universität Bayreuth, Universitätsstrasse 30, 95447 Bayreuth, Germany.
Lipps
Georg
G
Sugasawa
Kaoru
K
Iwai
Shigenori
S
Hanaoka
Fumio
F
Krauss
Gerhard
G
D020137:Base Pair Mismatch / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive / Q000502:physiology; D002945:Cisplatin / Q000737:chemistry; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D005454:Fluorescence Polarization / Q000379:methods; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D011233:Precipitin Tests / Q000379:methods; D013379:Substrate Specificity / Q000502:physiology; D051760:Xeroderma Pigmentosum Group A Protein
D018736:DNA Adducts; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C086984:XPC protein, human; D004247:DNA; D011088:DNA Ligases; D002945:Cisplatin