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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.
Insights
Researchers purified the XPE binding factor (XPE-BF), crucial for DNA repair in Xeroderma pigmentosum (XP) patients. This protein specifically targets UV-damaged DNA, aiding in genome surveillance and explaining XP group E
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Xeroderma pigmentosum (XP) is a genetic disorder impairing DNA repair after UV damage.
- A specific DNA binding factor, XPE-BF, is deficient in XP complementation group E patients.
- XPE-BF binds to UV-damaged DNA, cisplatin adducts, and denatured DNA.
Purpose of the Study:
- To purify and characterize the XPE binding factor (XPE-BF).
- To understand the binding properties and specificity of XPE-BF for damaged DNA.
- To elucidate the role of XPE-BF in the DNA repair pathway of Xeroderma pigmentosum.
Main Methods:
- Protein purification to near homogeneity.
- Gel filtration and glycerol gradient sedimentation for native protein analysis.
- DNA binding assays using UV-damaged and intact nucleotides.
- Novel 'shoe size' assay to determine minimal DNA substrate length.
Main Results:
- XPE-BF purified as a 125-kDa polypeptide, existing primarily as a monomer.
- Binding activity is dependent on cysteine residues, magnesium-stimulated, and zinc-inhibited.
- XPE-BF exhibits a 500,000-fold greater affinity for UV-damaged nucleotides compared to intact ones.
- Optimal binding requires a DNA substrate of 16-26 base pairs.
- XPE-BF binds to various UV lesions but may not bind to all cyclobutane dimers, including thymine dimers.
Conclusions:
- The purification and characterization of XPE-BF provide insights into its function in DNA repair.
- The high affinity for damaged DNA explains its role in genome surveillance despite low abundance.
- The differential binding to UV lesions may explain the milder phenotype observed in XP group E.
- These findings suggest the involvement of other DNA binding proteins in the XP repair pathway.