title: Transcription-coupled and DNA damage-dependent ubiquitination of RNA polymerase II in vitro.
abstract: Transcription-coupled repair (TCR) is essential for the rapid, preferential removal of DNA damage in active genes. The large subunit of RNA polymerase (Pol) II is ubiquitinated in cells after UV-irradiation or cisplatin treatment, which induces DNA damage preferentially repaired by TCR. Several human mutations, such as Cockayne syndrome complementation groups A and B, are defective in TCR and incapable of Pol II ubiquitination upon DNA damage. Here we demonstrate a correlation between ubiquitination of RNA Pol II and arrest of transcription in vitro. Ubiquitination of Pol II is significantly induced by alpha-amanitin, an amatoxin that blocks Pol II elongation and causes its degradation in cells. Pol II undergoes similar ubiquitination on DNA containing cisplatin adducts that arrest transcription. Stimulation of ubiquitination requires the addition of template DNA and does not occur in the presence of an antibody to the general transcription factor TFIIB, indicating the transcription dependence of the reaction. We propose that components of the reaction recognize elongating Pol II-DNA complexes arrested by alpha-amanitin or cisplatin lesions, triggering ubiquitination.
authors.affiliation: Center for Cancer Research and Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
authors.forename: Keng-Boon
authors.initials: KB
authors.lastname: Lee
authors.forename: Dong
authors.initials: D
authors.lastname: Wang
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Lippard
authors.forename: Phillip A
authors.initials: PA
authors.lastname: Sharp
mesh_terms: D000546:Amanitins / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage; D004260:DNA Repair; D006367:HeLa Cells; D006801:Humans; D012319:RNA Polymerase II / Q000378:metabolism*; D014158:Transcription, Genetic; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D000546:Amanitins; D025801:Ubiquitin; D012319:RNA Polymerase II; D002945:Cisplatin