Stable binding of human XPC complex to irradiated DNA confers strong discrimination for damaged sites.
Nucleotide excision repair (NER) of DNA damage requires an efficient means of discrimination between damaged and non-damaged DNA. Cells from humans with xeroderma pigmentosum group C do not perform NER in the bulk of the genome and are corrected by XPC protein, which forms a complex with hHR23B protein. This complex preferentially binds to some types of damaged DNA, but the extent of discrimination in comparison to other NER proteins has not been clear. Recombinant XPC, hHR23B, and XPC-hHR23B complex were purified. In a reconstituted repair system, hHR23B stimulated XPC activity tenfold. Electrophoretic mobility-shift competition measurements revealed a 400-fold preference for binding of XPC-hHR23B to UV damaged over non-damaged DNA. This damage preference is much greater than displayed by the XPA protein. The discrimination power is similar to that determined here in parallel for the XP-E factor UV-DDB, despite the considerably greater molar affinity of UV-DDB for DNA. Binding of XPC-hHR23B to UV damaged DNA was very fast. Damaged DNA-XPC-hHR23B complexes were stable, with half of the complexes remaining four hours after challenge with excess UV-damaged DNA at 30 degrees C. XPC-hHR23B had a higher level of affinity for (6-4) photoproducts than cyclobutane pyrimidine dimers, and some affinity for DNA treated with cisplatin and alkylating agents. XPC-hHR23B could bind to single-stranded M13 DNA, but only poorly to single-stranded homopolymers. The strong preference of XPC complex for structures in damaged duplex DNA indicates its importance as a primary damage recognition factor in non-transcribed DNA during human NER.
Batty
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Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire EN6 3LD, UK.
Rapic'-Otrin
V
V
Levine
A S
AS
Wood
R D
RD
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects*; D004260:DNA Repair / Q000235:genetics; D045643:DNA Repair Enzymes; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; C513287:RAD23B protein, human; D011994:Recombinant Proteins; C086985:RAD23A protein, human; C086984:XPC protein, human; D004247:DNA; D045643:DNA Repair Enzymes; D002945:Cisplatin