The human DNA repair gene, ERCC2 (XPD), corrects ultraviolet hypersensitivity and ultraviolet hypermutability of a shuttle vector replicated in xeroderma pigmentosum group D cells.
To determine the contribution of a human DNA repair gene, ERCC2 (XPD), to mutagenesis in human cells, two ERCC2 (XPD)-transformed xeroderma pigmentosum complementation group D (XPD) cell lines with increased UV survival compared to XP6BE(SV40), the original XPD line, were studied: D6BE-ER2-2 with slightly increased UV survival; and D6BE-ER2-9 with normal UV survival. ERCC2 (XPD) antibody-reactive protein levels were elevated 4.8-fold in D6BE-ER2-2 and 17.6-fold in D6BE-ER2-9 relative to XP6BE(SV40). DNA repair ability was assessed by measuring the ability of the cells to restore expression to UV-treated plasmids. Transfection of pRSVcat exposed to 1000 J/m2 UV resulted in 0.3% chloramphenicol acetyltransferase activity in XP6BE(SV40) cells but 20-80% in D6BE-ER2-2, D6BE-ER2-9, and repair-proficient cells compared to untreated control plasmids. The UV hypersensitivity of the mutagenesis shuttle vector pSP189 in XP6BE(SV40) cells was partially corrected and the UV hypermutability and excess of G:C-->A:T mutations of pSP189 fell to the normal range in D6BE-ER2-2 and D6BE-ER2-9 cells. However, the frequency of plasmids recovered with multiple base substitution mutations was significantly reduced with XP6BE(SV40) cells and remained low in D6BE-ER2-2 and D6BE-ER2-9 cells, when compared with the normal fibroblasts. The human DNA excision repair gene, ERCC2 (XPD), substantially corrected the plasmid UV hypersensitivity and UV hypermutability of xeroderma pigmentosum complementation group D cells; however, the dose response relationship varied for different end points.
Laboratory of Molecular Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892.
E M
EM
Gözükara
C N
CN
Parris
C A
CA
Weber
E P
EP
Salazar
M M
MM
Seidman
J F
JF
Watkins
L
L
Prakash
K H
KH
Kraemer
D000328:Adult; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011506:Proteins / Q000235:genetics*; D014157:Transcription Factors; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics*; D051759:Xeroderma Pigmentosum Group D Protein
D004268:DNA-Binding Proteins; D011506:Proteins; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein; C497292:ERCC2 protein, human