New link between the RNA polymerase II-CTD and replication stress.

Conflicts between transcription and replication are a major source of replication stress. Our recent findings show that proper dephosphorylation of Serine 5 in the carboxy-terminal domain (CTD) of DNA-directed RNA polymerase II subunit RPB1 is needed to prevent such conflicts in human cells.

WDR82/PNUTS-PP1 Prevents Transcription-Replication Conflicts by Promoting RNA Polymerase II Degradation on Chromatin.

Transcription-replication (T-R) conflicts cause replication stress and loss of genome integrity. However, the transcription-related processes that restrain such conflicts are poorly understood. Here, we demonstrate that the RNA polymerase II (RNAPII) C-terminal domain (CTD) phosphatase protein phosphatase 1 (PP1) nuclear targeting subunit (PNUTS)-PP1 inhibits replication stress. Depletion of PNUTS causes lower EdU uptake, S phase accumulation, and slower replication fork rates. In addition, the PNUTS binding partner WDR82 also promotes RNAPII-CTD dephosphorylation and suppresses replication stress. RNAPII has a longer residence time on chromatin after depletion of PNUTS or WDR82. Furthermore, the RNAPII residence time is greatly enhanced by proteasome inhibition in control cells but less so in PNUTS- or WDR82-depleted cells, indicating that PNUTS and WDR82 promote degradation of RNAPII on chromatin. Notably, reduced replication is dependent on transcription and the phospho-CTD binding protein CDC73 after depletion of PNUTS/WDR82. Altogether, our results suggest that RNAPII-CTD dephosphorylation is required for the continuous turnover of RNAPII on chromatin, thereby preventing T-R conflicts.

Regulation of ATR activity via the RNA polymerase II associated factors CDC73 and PNUTS-PP1.

Ataxia telangiectasia mutated and Rad3-related (ATR) kinase is a key factor activated by DNA damage and replication stress. An alternative pathway for ATR activation has been proposed to occur via stalled RNA polymerase II (RNAPII). However, how RNAPII might signal to activate ATR remains unknown. Here, we show that ATR signaling is increased after depletion of the RNAPII phosphatase PNUTS-PP1, which dephosphorylates RNAPII in its carboxy-terminal domain (CTD). High ATR signaling was observed in the absence and presence of ionizing radiation, replication stress and even in G1, but did not correlate with DNA damage or RPA chromatin loading. R-loops were enhanced, but overexpression of EGFP-RNaseH1 only slightly reduced ATR signaling after PNUTS depletion. However, CDC73, which interacted with RNAPII in a phospho-CTD dependent manner, was required for the high ATR signaling, R-loop formation and for activation of the endogenous G2 checkpoint after depletion of PNUTS. In addition, ATR, RNAPII and CDC73 co-immunoprecipitated. Our results suggest a novel pathway involving RNAPII, CDC73 and PNUTS-PP1 in ATR signaling and give new insight into the diverse functions of ATR.