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Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR)

A mouse model for the nucleotide excision repair disorder Cockayne syndrome (CS) was generated by mimicking a truncation in the CSB(ERCC6) gene of a CS-B patient. CSB-deficient mice exhibit all of the CS repair characteristics: ultraviolet (UV) sensitivity, inactivation of transcription-coupled repair, unaffected global genome repair, and inability to resume RNA synthesis after UV exposure. Other CS features thought to involve the functioning of basal transcription/repair factor TFIIH, such as growth failure and neurologic dysfunction, are present in mild form. In contrast to the human syndrome, CSB-deficient mice show increased susceptibility to skin cancer. Our results demonstrate that transcription-coupled repair of UV-induced cyclobutane pyrimidine dimers contributes to the prevention of carcinogenesis in mice. Further, they suggest that the lack of cancer predisposition in CS patients is attributable to a global genome repair process that in humans is more effective than in rodents.

Transcription‐coupled repair (TCR) is a universal sub‐pathway of the nucleotide excision repair (NER) system that is limited to the transcribed strand of active structural genes. It accomplishes the preferential elimination of transcription‐blocking DNA lesions and permits rapid resumption of the vital process of transcription. A defect in TCR is responsible for the rare hereditary disorder Cockayne syndrome (CS). Recently we found that mutations in the ERCC6 repair gene, encoding a putative helicase, underly the repair defect of CS complementation group B. Here we report the cloning and characterization of the Saccharomyces cerevisiae homolog of CSB/ERCC6, which we designate RAD26. A rad26 disruption mutant appears viable and grows normally, indicating that the gene does not have an essential function. In analogy with CS, preferential repair of UV‐induced cyclobutane pyrimidine dimers in the transcribed strand of the active RBP2 gene is severely impaired. Surprisingly, in contrast to the human CS mutant, yeast RAD26 disruption does not induce any UV‐, cisPt‐ or X‐ray sensitivity, explaining why it was not isolated as a mutant before. Recovery of growth after UV exposure was somewhat delayed in rad26. These findings suggest that TCR in lower eukaryotes is not very important for cell survival and that the global genome repair pathway of NER is the major determinant of cellular resistance to genotoxicity.

Transcription‐coupled repair (TCR), a subpathway of nucleotide excision repair (NER) defective in Cockayne syndrome A and B (CSA and CSB), is responsible for the preferential removal of DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. Here we demonstrate by microinjection of antibodies against CSB and CSA gene products into living primary fibroblasts, that both proteins are required for TCR and for recovery of RNA synthesis after UV damage in vivo but not for basal transcription itself. Furthermore, immunodepletion showed that CSB is not required for in vitro NER or transcription. Its central role in TCR suggests that CSB interacts with other repair and transcription proteins. Gel filtration of repair‐ and transcription‐competent whole cell extracts provided evidence that CSB and CSA are part of large complexes of different sizes. Unexpectedly, there was no detectable association of CSB with several candidate NER and transcription proteins. However, a minor but significant portion (10–15%) of RNA polymerase II was found to be tightly associated with CSB. We conclude that within cell‐free extracts, CSB is not stably associated with the majority of core NER or transcription components, but is part of a distinct complex involving RNA polymerase II. These findings suggest that CSB is implicated in, but not essential for, transcription, and support the idea that Cockayne syndrome is due to a combined repair and transcription deficiency.

Cockayne syndrome is a multisystem sun-sensitive genetic disorder associated with a specific defect in the ability to perform transcription-coupled repair of active genes after UV irradiation. Two complementation groups (CS-A and CS-B) have been identified, and 80% of patients have been assigned to the CS-B complementation group. We have analyzed the sites of the mutations in the CSB gene in 16 patients, to determine the spectrum of mutations in this gene and to see whether the nature of the mutation correlates with the type and severity of the clinical symptoms. In nine of the patients, the mutations resulted in truncated products in both alleles, whereas, in the other seven, at least one allele contained a single amino acid change. The latter mutations were confined to the C-terminal two-thirds of the protein and were shown to be inactivating by their failure to restore UV-irradiation resistance to hamster UV61 cells, which are known to be defective in the CSB gene. Neither the site nor the nature of the mutation correlated with the severity of the clinical features. Severe truncations were found in different patients with either classical or early-onset forms of the disease.

Cockayne syndrome (CS) is a nucleotide excision repair disorder characterized by sun (UV) sensitivity and severe developmental problems. Two genes have been shown to be involved: CSA and CSB. Both proteins play an essential role in preferential repair of transcriptionblocking lesions from active genes. In this study we report the purification and characterization of baculovirus-produced HA-His 6 -tagged CSB protein (dtCSB), using a highly efficient three-step purification protocol. Microinjection of dtCSB protein in CS-B fibroblasts shows that it is biologically functional in vivo. dtCSB exhibits DNA-dependent ATPase activity, stimulated by naked as well as nucleosomal DNA. Using structurally defined DNA oligonucleotides, we show that doublestranded DNA and double-stranded DNA with partial single-stranded character but not true single-stranded DNA act as efficient cofactors for CSB ATPase activity. Using a variety of substrates, no overt DNA unwinding by dtCSB could be detected, as found with other SNF2/ SWI2 family proteins. By site-directed mutagenesis the invariant lysine residue in the NTP-binding motif of CSB was substituted with a physicochemically related arginine. As expected, this mutation abolished ATPase activity. Surprisingly, the mutant protein was nevertheless able to partially rescue the defect in recovery of RNA synthesis after UV upon microinjection in CS-B fibroblasts. These results indicate that integrity of the conserved nucleotide-binding domain is important for the in vivo function of CSB but that also other properties independent from ATP hydrolysis may contribute to CSB biological functions.