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

© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.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.Norwegian Cancer Society [3367910]; South-Eastern Norway Health Authorities [2014035, 2013017]; Norwegian Research Council [275918]; EEA Czech-Norwegian Research Programme (Norwegian Financial Mechanism 2009–2014 and the Ministry of Education, Youth and Sports [Project Contract no. MSMT-22477/2014 (7F14061)]. Funding for open access charge: Norwegian Research Council.info:eu-repo/semantics/publishedVersio

SET8 is degraded via PCNA-coupled CRL4(CDT2) ubiquitylation in S phase and after UV irradiation

Degradation of the histone H4 methyltransferase SET8, which regulates chromosome compaction and genomic integrity, is regulated by the CRL4(CDT2) ubiquitin ligase to facilitate DNA replication and repair

Regulators of cyclin-dependent kinases are crucial for maintaining genome integrity in S phase

Maintenance of genome integrity is of critical importance to cells. To identify key regulators of genomic integrity, we screened a human cell line with a kinome small interfering RNA library. WEE1, a major regulator of mitotic entry, and CHK1 were among the genes identified. Both kinases are important negative regulators of CDK1 and -2. Strikingly, WEE1 depletion rapidly induced DNA damage in S phase in newly replicated DNA, which was accompanied by a marked increase in single-stranded DNA. This DNA damage is dependent on CDK1 and -2 as well as the replication proteins MCM2 and CDT1 but not CDC25A. Conversely, DNA damage after CHK1 inhibition is highly dependent on CDC25A. Furthermore, the inferior proliferation of CHK1-depleted cells is improved substantially by codepletion of CDC25A. We conclude that the mitotic kinase WEE1 and CHK1 jointly maintain balanced cellular control of Cdk activity during normal DNA replication, which is crucial to prevent the generation of harmful DNA lesions during replication

Simultaneous measurement of passage through the restriction point and MCM loading in single cells

Passage through the Retinoblastoma protein (RB1)-dependent restriction point and the loading of minichromosome maintenance proteins (MCMs) are two crucial events in G1-phase that help maintain genome integrity. Deregulation of these processes can cause uncontrolled proliferation and cancer development. Both events have been extensively characterized individually, but their relative timing and inter-dependence remain less clear. Here, we describe a novel method to simultaneously measure MCM loading and passage through the restriction point. We exploit that the RB1 protein is anchored in G1-phase but is released when hyper-phosphorylated at the restriction point. After extracting cells with salt and detergent before fixation we can simultaneously measure, by flow cytometry, the loading of MCMs onto chromatin and RB1 binding to determine the order of the two events in individual cells. We have used this method to examine the relative timing of the two events in human cells. Whereas in BJ fibroblasts released from G0-phase MCM loading started mainly after the restriction point, in a significant fraction of exponentially growing BJ and U2OS osteosarcoma cells MCMs were loaded in G1-phase with RB1 anchored, demonstrating that MCM loading can also start before the restriction point. These results were supported by measurements in synchronized U2OS cells

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