title: 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.
abstract: 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.
authors.affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.
authors.forename: Rebecca R
authors.initials: RR
authors.lastname: Selzer
authors.forename: Simon
authors.initials: S
authors.lastname: Nyaga
authors.forename: Jingsheng
authors.initials: J
authors.lastname: Tuo
authors.forename: Alfred
authors.initials: A
authors.lastname: May
authors.forename: Meltem
authors.initials: M
authors.lastname: Muftuoglu
authors.forename: Mette
authors.initials: M
authors.lastname: Christiansen
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Citterio
authors.forename: Robert M
authors.initials: RM
authors.lastname: Brosh
authors.forename: Vilhelm A
authors.initials: VA
authors.lastname: Bohr
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D002457:Cell Extracts; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D003057:Cockayne Syndrome / Q000201:enzymology / Q000235:genetics*; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics* / Q000528:radiation effects*; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair / Q000235:genetics*; D045643:DNA Repair Enzymes; D005347:Fibroblasts; D020869:Gene Expression Profiling; D005816:Genetic Complementation Test; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D000075223:Poly-ADP-Ribose Binding Proteins; D017434:Protein Structure, Tertiary; D012313:RNA / Q000096:biosynthesis; D011836:Radiation Tolerance / Q000235:genetics; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D002457:Cell Extracts; D000075223:Poly-ADP-Ribose Binding Proteins; C029389:thymine glycol; C024829:8-hydroxyguanine; D006147:Guanine; D012313:RNA; D003596:Cytosine; D006861:Hydrogen Peroxide; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C078673:ERCC6 protein, human; D045643:DNA Repair Enzymes; D013941:Thymine