No abstract

ion channel ͉ membrane protein ͉ structure ͉ acetylcholine

DNA is vulnerable to the attack of certain oxygen radicals and one of the major DNA lesions formed is 7,8-dihydro-8-oxoguanine (8-oxoG), a highly mutagenic lesion that can mispair with adenine. The repair of 8-oxoG was studied by measuring the gene specific removal of 8-oxoG after treatment of Chinese hamster ovary (CHO) fibroblasts with the photosensitizer Ro19-8022. This compound introduces 8-oxoG lesions, which can then be detected with the Escherichia coli formamidopyrimidine DNA glycosylase (FPG). In this report we present gene specific repair analysis of endogenous genes situated in different important cellular regions and also the first analysis of strand specific DNA repair of 8-oxoG in an endogenous gene. We were not able to detect any preferential repair of transcribed genes compared to non-transcribed regions and we did not detect any strand-bias in the repair of the housekeeping gene, dihydrofolate reductase (DHFR). In vivo, mitochondrial DNA is highly exposed to reactive oxygen species (ROS), and we find that the repair of 8-oxoG is more efficient in the mitochondrial DNA than in the nuclear DNA.

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein.The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and genespecific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV-or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways. INTRODUCTIONCockayne syndrome (CS) is an autosomal recessive human disorder with diverse clinical symptoms that include severe mental and physical growth retardation, microcephaly, progressive neurological and retinal degeneration, skeletal abnormalities, and a hypersensitivity to sunlight (Friedberg, 1996). Genetic analysis of fused heterodikaryons have identified two complementation groups involved in CS, designated CSA and CSB (Tanaka et al., 1981;Lehmann, 1982). CS cells demonstrate a reduced rate of nucleotide excision repair (NER) of active genes and, more specifically, of the transcribed strand of such genes (Venema et al., 1990;van Hoffen et al., 1993;Evans and Bohr, 1994). However, no defect in global NER is observed in CS. Defective transcription-coupled repair (TCR) in CS cells is found not only after UV exposure but also after exposure to certain forms of oxidative stress, suggesting that TCR is responsible for processing at least some types of oxidative DNA damage as well (Leadon and Cooper, 1993;Cooper and Leadon, 1994;Cooper et al., 1997).A characteristic feature of CS cells is the lack of recovery of RNA synthesis...