A High Through-Put Screen for Small Molecules Modulating MCM2 Phosphorylation Identifies Ryuvidine as an Inducer of the DNA Damage Response

Fitzgerald, Jennifer; Murillo, Laura S.; O'Brien, Gemma; O’Connell, Enda; O’Connor, Aisling; Wu, Kevin Z. L.; Wang, Guan‐Nan; Rainey, Michael D.; Natoni, Alessandro; Healy, Sandra; O’Dwyer, Michael; Santocanale, Corrado

doi:10.1371/journal.pone.0098891

Cited by 12 publications

(9 citation statements)

References 63 publications

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“…Depletion of endogenous RIF1 significantly increased the phosphorylation of chromatin‐associated MCM4, evident from its retardation on SDS–PAGE (Fig A (ii), lanes 2–4 and 6). Western analysis with phospho‐specific antibodies revealed increased phosphorylation of MCM2 at sites Ser40 and Ser53 (Fig A (ii), lower two panels), residues known to be targets of DDK . Note that phosphorylated MCM2 runs slightly faster in SDS–PAGE than the unphosphorylated form .…”

Section: Resultsmentioning

confidence: 96%

Human RIF 1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation

et al. 2017

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The human RIF1 protein controls DNA replication, but the molecular mechanism is largely unknown. Here, we demonstrate that human RIF1 negatively regulates DNA replication by forming a complex with protein phosphatase 1 (PP1) that limits phosphorylation‐mediated activation of the MCM replicative helicase. We identify specific residues on four MCM helicase subunits that show hyperphosphorylation upon RIF1 depletion, with the regulatory N‐terminal domain of MCM4 being particularly strongly affected. In addition to this role in limiting origin activation, we discover an unexpected new role for human RIF1‐PP1 in mediating efficient origin licensing. Specifically, during the G1 phase of the cell cycle, RIF1‐PP1 protects the origin‐binding ORC1 protein from untimely phosphorylation and consequent degradation by the proteasome. Depletion of RIF1 or inhibition of PP1 destabilizes ORC1, thereby reducing origin licensing. Consistent with reduced origin licensing, RIF1‐depleted cells exhibit increased spacing between active origins. Human RIF1 therefore acts as a PP1‐targeting subunit that regulates DNA replication positively by stimulating the origin licensing step, and then negatively by counteracting replication origin activation.

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Section: Resultsmentioning

confidence: 96%

Human RIF 1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation

et al. 2017

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“…HR deficiency leads to increase in single‐strand annealing , a key, nevertheless error‐prone, DSB repair pathway that results in the deletion of the repeat homologous nucleotide sequences generated during DSB formation . In an effort to improve the efficacy of the anti‐cancer therapies that target DNA, many have embarked on the quest for finding novel components of the DDR or compounds that specifically modulate the DNA repair mechanisms , aiming for a synergistic effect when administered with currently used DNA damaging drugs. Despite some initial roadblocks, some of the novel compounds found to inhibit molecules within DNA repair pathways have been successfully tested either in combination with present therapies or as stand‐alone treatments and are currently in clinical trials (Table ).…”

Section: Current Strategies Targeting Dna Replication and Dna Repairmentioning

confidence: 99%

Targeting DNA repair, DNA metabolism and replication stress as anti‐cancer strategies

2015

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Anti-cancer therapies targeting and damaging the DNA have been extensively used in the last 50 years since the discovery of nitrogen mustards, antimetabolites and platin agents. The use of these drugs is often limited by dose-limiting side effects related to their poor specificity. In recent years, much effort has been put on the discovery and development of compounds that would exploit defects in DNA repair in cancer cells such as Wee1, Chk1 or PARP1 inhibitors. However, not all cancers respond to these inhibitors. Recently, new developments towards specifically targeting broader characteristics of cancer such as replication stress (RS) and lost redox homeostasis have emerged. Oncogenes induce proliferation signals, which also result in replication-associated DNA damage, i.e. RS. Our knowledge into overall causes of RS, lesions produced and how these are signalled in cells to activate cell cycle checkpoints is evolving. Inhibition of ATR, which would normally keep non-deleterious levels of RS, induces intolerable RS levels for cancer cells. Interestingly, links between replication and transcription appear to underlie RS along with a reduction of the dNTP pool. Remarkably, sanitization of the dNTP pool by MutT homologue 1, impeding incorporation of oxidized dNTPs into the DNA, seems to be crucial for cancer cell survival. In this minireview we present an overview of current and novel strategies to target DNA repair and exploit DNA damage to treat cancer. We present the current models for cancer-associated RS as well as cancer phenotypic lethality. Both strategies are poised to better target cancer cells and reduce side effects.

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“…In particular CDC7 phosphorylation of MCM2 at Ser40 only occurs when Ser41 is also phosphorylated by a yet unidentified kinase, which acts as a priming event . While MCM2 Ser41 phosphorylation is constitutive, phosphorylation on Ser40 fluctuates during the cell-cycle strictly correlating with CDC7 activity, and it is considered a robust and reliable indicator or biomarker of cellular CDC7 activity. − CDC7 dependent phosphorylation of the MCM complex is counteracted by the protein phosphatase 1. − In S-phase, human CDC7 has other important functions, such as in the DNA replication stress response by phosphorylating the checkpoint mediator Claspin , and in the regulation of translesion DNA synthesis …”

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confidence: 99%

DNA Replication Dynamics and Cellular Responses to ATP Competitive CDC7 Kinase Inhibitors

et al. 2017

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The CDC7 kinase, by phosphorylating the MCM DNA helicase, is a key switch for DNA replication initiation. ATP competitive CDC7 inhibitors are being developed as potential anticancer agents; however how human cells respond to the selective pharmacological inhibition of this kinase is controversial and not understood. Here we have characterized the mode of action of the two widely used CDC7 inhibitors, PHA-767491 and XL-413, which have become important tool compounds to explore the kinase's cellular functions. We have used a chemical genetics approach to further characterize pharmacological CDC7 inhibition and CRISPR/CAS9 technology to assess the requirement for kinase activity for cell proliferation. We show that, in human breast cells, CDC7 is essential and that CDC7 kinase activity is formally required for proliferation. However, full and sustained inhibition of the kinase, which is required to block the cell-cycle progression with ATP competitor compounds, is problematic to achieve. We establish that MCM2 phosphorylation is highly sensitive to CDC7 inhibition and, as a biomarker, it lacks in dynamic range since it is easily lost at concentrations of inhibitors that only mildly affect DNA synthesis. Furthermore, we find that the cellular effects of selective CDC7 inhibitors can be altered by the concomitant inhibition of cell-cycle and transcriptional CDKs. This work shows that DNA replication and cell proliferation can occur with reduced CDC7 activity for at least 5 days and that the bulk of DNA synthesis is not tightly coupled to MCM2 phosphorylation and provides guidance for the development of next generation CDC7 inhibitors.

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A High Through-Put Screen for Small Molecules Modulating MCM2 Phosphorylation Identifies Ryuvidine as an Inducer of the DNA Damage Response

Cited by 12 publications

References 63 publications

Human RIF 1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation

Human RIF 1 and protein phosphatase 1 stimulate DNA replication origin licensing but suppress origin activation

Targeting DNA repair, DNA metabolism and replication stress as anti‐cancer strategies

DNA Replication Dynamics and Cellular Responses to ATP Competitive CDC7 Kinase Inhibitors

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