RNA polymerase II blockage by cisplatin-damaged DNA. Stability and polyubiquitylation of stalled polymerase.
The consequences of human RNA polymerase II (pol II) arrest at the site of DNA damaged by cisplatin were studied in whole cells and cell extracts, with a particular focus on the stability of stalled pol II and its subsequent ubiquitylation. Site-specifically platinated DNA templates immobilized on a solid support were used to perform in vitro transcription in HeLa nuclear extracts. RNA elongation was completely blocked by a cisplatin intrastrand cross-link. The stalled polymerase was quite stable, remaining on the DNA template in nuclear extracts. The stability of pol II stalled at the site of cisplatin damage was also observed in live cells. A cell fractionation experiment using cisplatin-treated HeLa cells revealed an increased level of chromatin-associated pol II proteins following DNA damage. The stalled polymerase was transcriptionally active and capable of elongating the transcript following chemical removal of platinum from the template. Transcription inhibition by alpha-amanitin in vitro enhanced pol II ubiquitylation at ubiquitin residues Lys-6, Lys-48, and Lys-63. In live cells expressing epitope-tagged ubiquitin mutants, several ubiquitin lysines also participated in pol II ubiquitylation following DNA damage. Cisplatin treatment triggered ubiquitylation-mediated pol II degradation in HeLa cells, which could be prevented by the proteasomal inhibitor MG132. Fractionation of pol II from cells co-treated with MG132 and cisplatin indicated that the undegraded ubiquitylated polymerase was mostly unbound or only loosely associated with chromatin. These data are consistent with a model in which only a fraction of pol II, ubiquitylated in response to cisplatin damage of DNA, dissociates from the sites of platination. This altered polymerase is rapidly destroyed by proteasomes.
Jung
Yongwon
Y
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307, USA.
Lippard
Stephen J
SJ
D002458:Cell Fractionation; D002467:Cell Nucleus / Q000201:enzymology; D004247:DNA / Q000187:drug effects* / Q000494:pharmacology*; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000138:chemical synthesis / Q000187:drug effects; D004795:Enzyme Stability; D006367:HeLa Cells; D006801:Humans; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors*; D014158:Transcription, Genetic / Q000187:drug effects; D025801:Ubiquitin / Q000378:metabolism*
D004277:DNA, Single-Stranded; D025801:Ubiquitin; D004247:DNA; D012319:RNA Polymerase II