Phenotypic consequences of mutations in the conserved motifs of the putative helicase domain of the human Cockayne syndrome group B gene.
Cockayne syndrome (CS) is a human genetic disorder characterized by several neurological and developmental abnormalities. Two genetic complementation groups, CS-A and CS-B, have been identified. The CSB protein belongs to helicase superfamily 2, and to the SWI/SNF family of proteins. The CSB protein is implicated in transcription-coupled repair (TCR), basal transcription and chromatin remodeling. In addition, CS cells undergo UV-induced apoptosis at much lower doses than normal cells. However, the molecular function of the CSB protein in these biological pathways has remained unclear. Evidence indicates that the integrity of the Walker A and B boxes (motifs I and II) are important for CSB function, but the functional significance of the helicase motifs Ia, III--IV has not been previously examined. In this study, single amino acid changes in highly conserved residues of helicase motifs Ia, III, V, VI and a second putative nucleotide-binding motif (NTB) of the CSB protein were generated by site-directed mutagenesis to analyze the genetic function of the CSB protein in survival, RNA synthesis recovery and apoptosis after UV treatment. The survival analysis of these CS-B mutant cell lines was also performed after treatment with the chemical carcinogen, 4-nitroquinoline-1-oxide (4-NQO). The lesions induced by UV light, cyclobutane pyrimidine dimers, are known to be repaired by TCR whereas the lesions induced by 4-NQO are repaired by global genome repair. The results of this study demonstrate that the point mutations in highly conserved residues of helicase motifs Ia, III, V and VI abolished the genetic function of the CSB protein in survival, RNA synthesis recovery and apoptosis after UV treatment. Similarly, the same mutants failed to complement the sensitivity toward 4-NQO. Thus, the integrity of these helicase motifs is important for the biological function of the CSB protein. On the contrary, a point mutation in a C-terminal, second, NTB motif of the CSB protein showed full complementation in the ability to repair damage induced by UV light or 4-NQO, suggesting that this motif is not important for the CSB repair function.
Muftuoglu
Meltem
M
Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.
Selzer
Rebecca
R
Tuo
Jingsheng
J
Brosh
Robert M
RM
Bohr
Vilhelm A
VA
D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology; D020816:Amino Acid Motifs / Q000235:genetics*; D000595:Amino Acid Sequence; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D017124:Conserved Sequence / Q000235:genetics; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D045643:DNA Repair Enzymes; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D005786:Gene Expression Regulation / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D010641:Phenotype; D000075223:Poly-ADP-Ribose Binding Proteins; D015363:Quinolones / Q000494:pharmacology; D012313:RNA / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D014466:Ultraviolet Rays
C120195:4-nitroquinolone-1-oxide; D000075223:Poly-ADP-Ribose Binding Proteins; D015363:Quinolones; D015112:4-Nitroquinoline-1-oxide; D012313:RNA; D004265:DNA Helicases; C078673:ERCC6 protein, human; D045643:DNA Repair Enzymes