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.

Additional Metadata
Keywords *DNA Repair, *Transcription, Genetic, 0 (CKN1 protein, human), 0 (Macromolecular Systems), 0 (Proteins), 148972-58-1 (ERCC6 protein), Cockayne Syndrome/*genetics, Consensus Sequence, DNA Helicases/genetics/metabolism/*physiology, EC 2.7.7.- (RNA Polymerase II), EC 5.99.- (DNA Helicases), Hela Cells, Human, Macromolecular Systems, Proteins/metabolism, RNA Polymerase II/*metabolism, Support, Non-U.S. Gov't
Persistent URL hdl.handle.net/1765/3122
Citation
van Gool, A.J., Citterio, E., Rademakers, S., van Os, R., Vermeulen, W., Constantinou, A., … Hoeijmakers, J.H.J.. (1997). The Cockayne syndrome B protein, involved in transcription-coupled repair resides in a RNA polymerase II-containing complex.. EMBO Journal, 16, 5955–5965. Retrieved from http://hdl.handle.net/1765/3122