Purification and characterization of a human protein that binds to damaged DNA.
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.
Hwang
B J
BJ
Department of Medicine, Stanford University Medical Center, California 94305.
Chu
G
G
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
D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA; D008274:Magnesium; D015032:Zinc; D002945:Cisplatin