title: Purification and characterization of a human protein that binds to damaged DNA.
abstract: Xeroderma pigmentosum (XP) is an inherited disease characterized by defective repair of DNA damaged by ultraviolet (UV) radiation or agents that produce bulky DNA adducts. Human cells contain a factor that is deficient in a subset of patients from XP complementation group E and binds to DNA damaged by UV, cisplatin, or denaturation. This factor, XPE binding factor (XPE-BF), was purified to near homogeneity. The denatured protein migrated as a 125-kDa polypeptide on SDS-PAGE, and the native protein migrated primarily as a monomer on gel filtration and glycerol gradient sedimentation. Sedimentation revealed major peak in binding activity at 6.8 S, corresponding to the monomeric form, and a minor peak at 14.5 S, suggesting a homodimeric form. Binding activity was dependent on unmodified cysteine residues, stimulated by magnesium, and inhibited by zinc. Binding to UV-damaged nucleotides was 500,000-fold greater than for intact nucleotides, explaining how a molecule with an abundance of only 1-2 molecules per megabase can survey the genome for damaged DNA. Binding required a minimal DNA substrate of between 16 and 26 bp, as determined by a novel "shoe size" assay. Consistent with its previously noted versatility, XPE-BF bound to some cyclobutane pyrimidine dimers and at least one other UV-induced lesion. However, it may not bind to a subset of cyclobutane dimers, likely including the thymine dimer. These findings may explain the relatively mild phenotype of XP group E and suggest the existence of at least one other binding protein involved in the XP repair pathway.
authors.affiliation: Department of Medicine, Stanford University Medical Center, California 94305.
authors.forename: B J
authors.initials: BJ
authors.lastname: Hwang
authors.forename: G
authors.initials: G
authors.lastname: Chu
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D004249:DNA Damage; D005260:Female; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011489:Protein Denaturation; D013379:Substrate Specificity; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology
chemical_list: D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA; D008274:Magnesium; D015032:Zinc; D002945:Cisplatin