Fate of RNA polymerase II stalled at a cisplatin lesion - Archive ouverte HAL
Journal Articles Journal of Biological Chemistry Year : 2004

Fate of RNA polymerase II stalled at a cisplatin lesion

Abstract

Elongating RNA polymerase II blocked by DNA damage in the transcribed DNA strand is thought to initiate the transcription-coupled repair process. The objective of this study is to better understand the sequence of events that occurs during repair from the time RNA polymerase II first encounters the lesion. This study establishes that an immobilized DNA template containing a unique cisplatin lesion can serve as an in vitro substrate for both transcription and DNA repair. RNA polymerase II is quantitatively stalled at the cisplatin lesion during transcription and can be released from the template, along with the nascent transcript, in an ATP-dependent manner. RNA polymerase II stalled at a lesion and containing a dephosphorylated repetitive carboxyl-terminal domain (CTD) appears to be more sensitive toward release. However, a dephosphorylated CTD can become readily phosphorylated in front of the lesion by CTD kinases in the presence of ATP. The observation that RNA polymerase II and transcript release occurs in a TFIIH-deficient repair extract but not in a reconstituted repair system demonstrates that disassembly of the elongation complex can occur independently of the repair process and vice versa. Indeed, the presence of RNA polymerase II at the lesion does not prevent dual incision from occurring. Finally, we also propose that the Cockayne's syndrome B protein factor, believed to be the mammalian transcription repair coupling factor, is neither involved in transcript release nor required for dual incision in the presence of lesionstalled RNA polymerase II in vitro. More likely, it prevents RNA polymerase from backing up when it encounters the lesion. The ability to transcribe and repair the same damaged DNA molecule fixed on beads, along with the fact that the reaction conditions can be freely altered, provides a powerful tool to study the fate of RNA polymerase II blocked on the cisplatin lesion.

Dates and versions

hal-04127594 , version 1 (13-06-2023)

Identifiers

Cite

Alexandre Tremeau-Bravard, Thilo Riedl, Jean-Marc Egly, Michael E. Dahmus. Fate of RNA polymerase II stalled at a cisplatin lesion. Journal of Biological Chemistry, 2004, 279 (9), pp.7751-7759. ⟨10.1074/jbc.M309853200⟩. ⟨hal-04127594⟩
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