The Cockayne syndrome B protein, involved in transcription-coupled DNA repair, resides in an RNA polymerase II-containing complex.
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.
van Gool
A J
AJ
MGC Department of Cell Biology and Genetics, Erasmus University Rotterdam, PO Box 1738, 3000 DR, Rotterdam, The Netherlands.
Citterio
E
E
Rademakers
S
S
van Os
R
R
Vermeulen
W
W
Constantinou
A
A
Egly
J M
JM
Bootsma
D
D
Hoeijmakers
J H
JH
D003057:Cockayne Syndrome / Q000235:genetics*; D016384:Consensus Sequence; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004260:DNA Repair; D045643:DNA Repair Enzymes; D006367:HeLa Cells; D006801:Humans; D046911:Macromolecular Substances; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic
C095472:ERCC8 protein, human; D046911:Macromolecular Substances; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins; D014157:Transcription Factors; D012319:RNA Polymerase II; D004265:DNA Helicases; C078673:ERCC6 protein, human; D045643:DNA Repair Enzymes