RNA Polymerase II Blockage by Cisplatin-damaged DNA: STABILITY AND POLYUBIQUITYLATION OF STALLED POLYMERASE

Yongwon Jung,Stephen J Lippard

doi:10.1074/jbc.m509688200

Yongwon Jung, Stephen J Lippard

Open Access

https://doi.org/10.1074/jbc.m509688200

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Abstract

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.

Highlights

The anti-tumor activity of platinum-based drugs is mediated by their ability to attack DNA
Stability of polymerase II (pol II) Stalled at a Platinum-DNA Lesion—A linear DNA template containing an adenovirus major late (AdML) promoter and a cisplatin-DNA lesion with a biotin moiety was prepared (Fig. 1)
Most 114-nt transcripts were found on the beads, indicating that pol II stalled at a cisplatin lesion is fairly stable under these conditions

Summary

Introduction

The anti-tumor activity of platinum-based drugs is mediated by their ability to attack DNA. Damage-specific pol II proteins (which were tightly bound to DNA and remained on the beads after a 1% Sarkosyl wash) appeared only in the transcription reaction with cisplatin-damaged templates in the absence of ␣-amanitin (Fig. 4B, lanes 3, 6, and 12 versus lane 9).

Results

Conclusion