DNAPKcs-dependent arrest of RNA polymerase II transcription in the presence of DNA breaks

Abstract

DNA double-strand break (DSB) repair interferes with ongoing cellular processes, including replication and transcription. Although the process of replication stalling upon collision of replication forks with damaged DNA has been extensively studied, the fate of elongating RNA polymerase II (RNAPII) that encounters a DSB is not well understood. We show that the occurrence of a single DSB at a human RNAPII-transcribed gene leads to inhibition of transcription elongation and reinitiation. Upon inhibition of DNA protein kinase (DNAPK), RNAPII bypasses the break and continues transcription elongation, suggesting that it is not the break per se that inhibits the processivity of RNAPII, but the activity of DNAPK. We also show that the mechanism of DNAPK-mediated transcription inhibition involves the proteasome-dependent pathway. The results point to the pivotal role of DNAPK activity in the eviction of RNAPII from DNA upon encountering a DNA lesion.

The emerging role of Polycomb repressors in the response to DNA damage

“…The physiological relevance of these findings has recently been supported by ChIP-based studies that mapped the distribution of both cH2AX and Pol II in parallel with transcription occurring around endonuclease-induced breaks (Iacovoni et al, 2010;Pankotai et al, 2012). Importantly, the occurrence of a single DSB in the body of genes transcribed by Pol II results in inhibition of transcription (Iacovoni et al, 2010;Pankotai et al, 2012). The interplay between DDR and transcription could have more than one purpose.…”

“…The existence of this phenomenon was demonstrated using stably integrated constructs that allow the induction of a defined DNA break in the direct vicinity of a transcriptional reporter gene (Cuozzo et al, 2007;O'Hagan et al, 2008;. The physiological relevance of these findings has recently been supported by ChIP-based studies that mapped the distribution of both cH2AX and Pol II in parallel with transcription occurring around endonuclease-induced breaks (Iacovoni et al, 2010;Pankotai et al, 2012). Importantly, the occurrence of a single DSB in the body of genes transcribed by Pol II results in inhibition of transcription (Iacovoni et al, 2010;Pankotai et al, 2012).…”

Nuclear position dictates DNA repair pathway choice

“…RNA was extracted using the RNeasy minikit (Qiagen) according to the manufacturer's protocol. RT-qPCRs were then processed as in (Pankotai et al 2012). Proteins were extracted in RIPA buffer and analyzed by Western blot.…”

ATR checkpoint kinase and CRL1βTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks

“…The kinase activity of ATM also inhibits RNA polymerase I following DSBs (Kruhlak et al 2007). Inhibition of RNA polymerase II-dependent gene transcription following I-PpoI-induced DNA breakage is dependent on DNA-PKcs (Pankotai et al 2012). Although we expected clusters of stalled forks to give rise to doublestrand ends and activate ATM and DNA-PKcs, the checkpoint pathway reported here is exclusively mediated by ATR but not ATM and DNA-PKcs.…”

“…ultimately leading to arrest of the cell cycle in S phase. Since transcription of neighboring genes is often repressed by even a single DNA double-strand break (DSB) in mammalian cells by active pathways using ATM and DNA-dependent protein kinases (Kruhlak et al 2007;Shanbhag et al 2010;Pankotai et al 2012), we wondered whether and how stalled replication forks produced by anticancer drugs affect the transcription of neighboring genes. Doxorubicin (DOX) is a widely used cancer chemotherapeutic drug that works by intercalating into dsDNA, inhibiting the activity of DNA topoisomerase II (Bodley et al 1989;Capranico et al 1990).…”