Nucleic Acids Research, 2002, Vol. 30, No. 3 782-793
© 2002 Oxford University Press
Differential requirement for the ATPase domain of the Cockayne syndrome group B gene in the processing of UV-induced DNA damage and 8-oxoguanine lesions in human cells
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA, 1Department of Biochemistry, School of Medicine, Hacettepe University, Ankara, Turkey, 2Danish Center for Molecular Gerontology, Department of Molecular and Structural Biology, University of Aarhus, DK-8000 Aarhus C, Denmark and 3Medical Genetics Centre, Department of Cell Biology and Genetics, Erasmus University Rotterdam, Rotterdam, The Netherlands
Cockayne syndrome (CS) is a rare inherited human genetic disorder characterized by UV sensitivity, developmental abnormalities and premature aging. The cellular and molecular phenotypes of CS include increased sensitivity to oxidative and UV-induced DNA lesions. The CSB protein is thought to play a pivotal role in transcription-coupled repair and CS-B cells are defective in the repair of the transcribed strand of active genes, both after exposure to UV and in the presence of oxidative DNA lesions. A previous study has indicated that a conserved helicase ATPase motif II residue is essential for the function of the CSB protein in responding to UV-induced DNA damage in a hamster cell line. Due to the limitations in studying a complex human disorder in another species, this study introduced the site-directed mutation of the ATPase motif II in the human CSB gene in an isogenic human cell line. The CSB mutant allele was tested for genetic complementation of UV-sensitive phenotypes in the human CS-B cell line CS1AN.S3.G2. In addition, the incision of an 8-oxoguanine lesion by extracts of the CS-B cell lines stably transfected with the wild-type or ATPase mutant CSB gene has been investigated. The ATPase motif II point mutation (E646Q) abolished the function of the CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery and apoptosis. Interestingly, whole-cell extract prepared from these mutant cells retained wild-type incision activity on an oligonucleotide containing a single 8-oxoguanine lesion, whereas the absence of the CSB gene altogether resulted in reduced incision activity relative to wild-type. These results suggest damage-specific functional requirements for CSB in the repair of UV-induced and oxidative lesions in human cells. The transfection of the mutant or wild-type CSB gene into the CS1AN.S3.G2 cells did not alter the expression of the subset of genes examined by cDNA array analysis.
* To whom correspondence should be addressed. Tel: +1 410 558 8162; Fax: +1 410 558 8157; Email: vbohr{at}nih.gov Present address: Rebecca R. Selzer, Department of Anesthesiology and Waisman Center on Mental Retardation and Development, University of Wisconsin-Madison, Madison, WI 53705, USA
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