Analysis of cells harboring a putative DNA repair gene reveals a lack of evidence for a second independent xeroderma pigmentosum group A correcting gene.
The UV hypersensitivity of xeroderma pigmentosum (XP) complementation group A cells is restored to near-normal by transfection of the XPA gene located on human chromosome 9. However, it has been reported that a cosmid related to a cDNA on chromosome 8 is also able to partially correct the UV sensitivity of XP-A cells. We describe here an investigation of a representative cosmid transfectant, denoted 2-0-A2. Whole cell extracts prepared from 2-0-A2 cells carried out DNA repair synthesis in vitro that was in the normal range, consistent with their UV-resistant phenotype. Immunoblotting indicated that 2-0-A2 cells expressed full-length XPA protein. This was unexpected because the 2-0-A2 cell line was thought to have been isolated by transfection of a cell line derived from patient XP2OS, and a known homozygous mutation in XP2OS prevents expression of XPA gene product. This mutation creates an AlwNI restriction endonuclease cleavage site in XPA and was not present in 2-0-A2. These results prompted an RFLP analysis which revealed that the 2-0-A2 cell line was not derived from XP2OS but from another line that fails to express XPA protein, XP12BE. It appears that the significant UV-resistance and DNA repair capacity of 2-0-A2 can be ascribed to the re-expression of XPA in XP12BE, and it is unnecessary to postulate a second XP-A complementing gene to explain the results.
Jones
C J
CJ
Department of Pathology, University of Wales College of Medicine, Heath Park, Cardiff, UK.
Lloyd
R S
RS
Wood
R D
RD
D001483:Base Sequence; D002460:Cell Line; D002474:Cell-Free System; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D051760:Xeroderma Pigmentosum Group A Protein
D004268:DNA-Binding Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C053688:endodeoxyribonuclease AlwNI; D015252:Deoxyribonucleases, Type II Site-Specific