Transcription-coupled and DNA damage dependent ubiquitination of RNA polymerase II in vitro

Transcription-coupled repair (TCR) is essential for the rapid, preferential removal of DNA damage in active genes. The large subunit of RNA polymerase (Pol) ll 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 11 and arrest of transcription in vitro. Ubiquitination of Pol II is significantly induced by a-amanitin, an amatoxin that blocks Pol 11 elongation and causes its degradation in cells. Pol 11 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 TFIIe, indicating the transcription dependence of the reaction. We propose that components of the reaction recognize elongating Pol 11-DNA complexes arrested by a-amanitin or cisplatin lesions; triggering ubiquitination.