Identification of ICR170-induced XPD mutations in UV-sensitive CHO cells.
We highlight selected contributions of Dr. Richard Setlow that contributed to our earlier understanding of excision repair processes and set the stage for dissecting nucleotide excision repair (NER) in mammalian cells through molecular genetics. More than 20 years ago, large-scale screens for UV-sensitive mutants of hamster CHO cells isolated approximately 200 mutants, many of which were assigned to the XPD/ERCC2 complementation group, but the nature of the mutations was not determined. The XPD protein performs not only an essential viability function as a structural component of transcription initiation factor TFIIH, but also an NER function as a 5' to 3' DNA helicase within TFIIH that unwinds DNA on the 3' side of bulky lesions. Alterations in these XPD functions are responsible for three UV-sensitivity genetic disorders that have distinguishable clinical features. In this study, we sequenced six UV-sensitive ICR170-induced Chinese hamster ovary (CHO) cell mutants that previously were assigned to the XPD complementation group to determine whether they carry frameshift mutations. All six mutants show 3- to 5-fold increased hypersensitivity to UV irradiation, similar to the XPD mutant prototype UV5. Even though ICR170 is a strong frameshift mutagen, all six cell lines contain base substitution mutations, five of which are unique among all mutations identified so far in human and rodent cells. The sixth mutation was identical to the R75W mutation previously found in CHO UVL-1. The results presented here contribute to a mutation database that should prove useful in structure-function studies of this unique DNA-structure-specific helicase and its complex mutant phenotypes.
Tebbs
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RS
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551-0808, USA.
Salazar
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EP
Thompson
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LH
D000609:Aminoacridines / Q000633:toxicity*; D000818:Animals; D016466:CHO Cells / Q000502:physiology* / Q000528:radiation effects; D006224:Cricetinae; D004265:DNA Helicases; D004260:DNA Repair; D004268:DNA-Binding Proteins; D009153:Mutagens / Q000633:toxicity*; D009154:Mutation; D009588:Nitrogen Mustard Compounds / Q000633:toxicity*; D011506:Proteins / Q000235:genetics*; D011836:Radiation Tolerance / Q000235:genetics; D014157:Transcription Factors; D014466:Ultraviolet Rays; D051759:Xeroderma Pigmentosum Group D Protein
D000609:Aminoacridines; D004268:DNA-Binding Proteins; D009153:Mutagens; D009588:Nitrogen Mustard Compounds; D011506:Proteins; D014157:Transcription Factors; C091910:acridine mustard; D004265:DNA Helicases; D051759:Xeroderma Pigmentosum Group D Protein