of work: Cockayne syndrome (CS) belongs to the category of premature aging diseases, where the individuals appear much older than their chronological age. Cells from CS patients are sensitive to UV light, exhibit a delay in recovery of DNA and RNA synthesis following irradiation, and are defective in preferential repair and strand-specific repair of active genes. Complementation studies demonstrate at least two genes involved in CS, designated CSA and CSB. CSB protein, by sequence comparison, belongs to the SNF2 family of proteins, which have roles in transcriptional regulation, chromosome stability and DNA repair. The cellular and molecular phenotype of CS include a significantly increased sensitivity to a number of DNA-damaging agents including UV irradiation. Studies in CS cells were initially confined to DNA repair in the general, overall genome, where no defect was found. However, CS cells are defective in the preferential repair of active genes and in the preferential repair of the transcribed strand of such genes. Transfection of the CSB gene into hamster cells with the CS-B phenotype completely restores TCR and UV resistance to normal levels, demonstrating that the defect in TCR in CS-B is due to mutation in that gene. The complex clinical phenotype of CS, however, suggests that DNA repair may not be the only defect. We have reported a defect in basal transcription in CS both in vivo and in vitro. This transcription defect is seen in CS-B lymphoblastoid cells and fibroblasts without any exposure to stress such as UV light. We have used an in vitro assay to measure the incision event of the DNA repair process. During the first step of BER, there is an incision in DNA 5' to the lesion. The incision can be quantitated in cell extracts by using oligonucleotide duplexes that contain a single 8-oxoG and 8-oxoG lesion at a defined site. In CS-B deficient cell lines we observe a decrease in 8-oxoG incision that can be complemented by transfection of a plasmid containing the intact CSB gene. This suggests a direct role for CSB in the recognition of oxidative DNA damage. The CSB protein apparently functions at the crossroads of DNA repair and transcription. We have also observed that CS-B cells appear to have a more open chromatin structure than normal cells, what would be compatible with a role in chromatin structural assembly. It would appear that the CSB protein has more than one function and is probably involved in a large number of protein-protein interactions in transcription and repair pathways. One or more of these is likely to be very important for the assembly of the DNA repair and transcription complexes at the nuclear matrix. A functional analysis of the CSB gene has been undertaken in our laboratory to better understand the nature of the molecular deficiencies observed in CS. Mutants, generated by site- directed mutagenesis, have been tested for genetic complementation of CSB null cell lines by cell viability and RNA synthesis recovery upon exposure to UV light and other genotoxic agents. Point mutations in ATPase motifs I and II of CSB dramatically reduce CSB function in vivo, suggesting that ATP hydrolysis by CSB protein is required for transcription-coupled repair of DNA damage. This mutant also shows dramatically increased apoptosis, suggesting a role for the CS protein in the apoptotic pathway. We have now made a series of point mutations in the helicase domain of the CSB gene in stably transfected human cell lines. We find that some of these are defective in the incision of 8-oxoG, suggesting a defect in base excision repair. Further, we have analyzed the formation of another important oxidative DNA base lesion, 8-hydroxyadenine. The repair of this adduct is also deficient in CSB. We can conclude that the CSB protein is involved in the general genome base excision repair process, and that different domains of the helicase region play different roles in this process. We find that the repair defect that we observe directly correlates with cellular sensitivity to X-ray and that oxidative lesions accumulate in CSB cells after exposure. This could explain the high prevalence of neurological defects seen in CSB patients. The repair defect in CSB cells is not limited to the nuclear DNA, but also observed in mitochondrial DNA. Thus, remaining lesions in mitochondrial DNA in patients might contribute to the aging and neurodegenerative phenotypes. We have studied the function and protein interactions of the purified CSB protein. We have characterized its ATPase activity and find that it is subject to phosphorylation. The CSB protein inetracts with some of the proteins that are involved in the DNA repair of oxidative damage, the process called base excision repair. This does suggest that proficient base excision repair requires a functional CSB protein.
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