Isolation of a cDNA encoding a UV-damaged DNA binding factor defective in xeroderma pigmentosum group E cells
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1Department of Medicine, Stanford University Medical Center, CA 94305, USA.
Abstract:
XPE binding factor (XPE-BF) is deficient in a subset of patients from xeroderma pigmentosum complementation group E. Binding activity copurifies with a 125 kDa polypeptide (p125) that binds to DNA damaged by ultraviolet (UV) radiation and many other agents. We isolated cDNA encoding a polypeptide with a predicted amino acid sequence that matched the sequences of eleven tryptic peptides derived from digestion of XPE-BF purified from human placenta. In vitro transcription and translation of the cDNA yielded a polypeptide of 125 kDa that bound specifically to UV-damaged DNA. Therefore the cDNA encodes either all or the major component of XPE-BF.
Insights
Researchers identified the gene for XPE binding factor (XPE-BF), crucial for DNA repair in xeroderma pigmentosum patients. This discovery advances understanding of DNA damage response and genetic disorders.
Area of Science:
- Molecular biology
- Genetics
- DNA repair mechanisms
Background:
- Xeroderma pigmentosum (XP) is a genetic disorder characterized by extreme sensitivity to sunlight.
- Deficiency in XPE binding factor (XPE-BF) is observed in a subset of xeroderma pigmentosum complementation group E patients.
- XPE-BF is known to bind to DNA damaged by ultraviolet (UV) radiation and other genotoxic agents.
Purpose of the Study:
- To isolate and characterize the gene encoding the XPE binding factor (XPE-BF).
- To confirm the identity and function of the polypeptide produced by the isolated cDNA.
Main Methods:
- Purification of XPE-BF from human placenta.
- Peptide sequencing of the purified protein.
- Isolation of cDNA encoding the XPE-BF polypeptide.
- In vitro transcription and translation of the cDNA.
- DNA binding assays using the synthesized polypeptide.
Main Results:
- A 125 kDa polypeptide (p125) copurifying with XPE-BF was identified as the DNA-binding component.
- cDNA encoding a polypeptide matching the tryptic peptides of p125 was isolated.
- In vitro synthesized p125 specifically bound to UV-damaged DNA.
- The isolated cDNA likely encodes the major component or the entirety of XPE-BF.
Conclusions:
- The isolated cDNA encodes the primary component of XPE binding factor.
- This finding contributes to understanding the molecular basis of xeroderma pigmentosum complementation group E.
- The identified gene is critical for DNA repair pathways involved in protecting against UV-induced damage.