Direct Role for the Replication Protein Treslin (Ticrr) in the ATR Kinase-mediated Checkpoint Response

Hassan, Bachar H.; Lindsey-Boltz, Laura A.; Kemp, Michael G.; Sancar, Aziz

doi:10.1074/jbc.m113.475517

Cited by 16 publications

(20 citation statements)

References 31 publications

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“…25,26,[32][33][34][35][36] However, we observed that though the complex is stable in buffers of high ionic strength, it precipitated under reaction conditions necessary to measure kinase activity. Thus we were unable to test the contribution of RHINO to ATR-Chk1 signaling in vitro.…”

Section: Rhino Directly Binds To Topbp1mentioning

confidence: 42%

“…33,40,41 We therefore made use of this methodology to further validate RHINO as a bona fide mediator of ATR-Chk1 signaling in human cells. We thus fused the cDNA encoding the Lac repressor (LacR) to the RHINO cDNA and then introduced the construct into NIH3T3 cells containing hundreds of repeats of the LacO DNA sequence.…”

Section: Tethering Of Rhino To Chromatin Activates Atr-chk1 Signalingmentioning

confidence: 99%

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RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling

et al. 2015

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The ATR-Chk1 signaling pathway mediates cellular responses to DNA damage and replication stress and is composed of a number of core factors that are conserved throughout eukaryotic organisms. However, humans and other higher eukaryotic species possess additional factors that are implicated in the regulation of this signaling network but that have not been extensively studied. Here we show that RHINO (for Rad9, Rad1, Hus1 interacting nuclear orphan) forms complexes with both the 9-1-1 checkpoint clamp and TopBP1 in human cells even in the absence of treatments with DNA damaging agents via direct interactions with the Rad9 and Rad1 subunits of the 9-1-1 checkpoint clamp and with the ATR kinase activator TopBP1. The interaction of RHINO with 9-1-1 was of sufficient affinity to allow for the purification of a stable heterotetrameric RHINO-Rad9-Hus1-Rad1 complex in vitro. In human cells, a portion of RHINO localizes to chromatin in the absence of DNA damage, and this association is enriched following UV irradiation. Furthermore, we find that the tethering of a Lac Repressor (LacR)-RHINO fusion protein to LacO repeats in chromatin of mammalian cells induces Chk1 phosphorylation in a Rad9-and Claspin-dependent manner. Lastly, the loss of RHINO partially abrogates ATR-Chk1 signaling following UV irradiation without impacting the interaction of the 9-1-1 clamp with TopBP1 or the loading of 9-1-1 onto chromatin. We conclude that RHINO is a bona fide regulator of ATR-Chk1 signaling in mammalian cells.

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Section: Rhino Directly Binds To Topbp1mentioning

confidence: 42%

Section: Tethering Of Rhino To Chromatin Activates Atr-chk1 Signalingmentioning

confidence: 99%

RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling

et al. 2015

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“…In humans, partial reconstitution reactions have been reported with ATR-ATRIP ϩ DNA (5); ATR-ATRIP ϩ ssDNA ϩ RPA (22); and ATR-ATRIP ϩ TopBP1 ϩ ssDNA ϩ RPA Ϯ Claspin with substrates that included RPA, Chk1, and p53 (23,29,37,(61)(62)(63)(64). In addition to these systems with purified proteins, a number of other in vitro systems with cell-free extracts have been reported (28, 34 -36, 65).…”

Section: Discussionmentioning

confidence: 99%

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

Lindsey-Boltz

Kemp

Reardon

et al. 2014

Journal of Biological Chemistry

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Background: Nucleotide excision repair and the ATR-mediated DNA damage checkpoint responses are genetically coupled. Results: We have analyzed the basic steps of ATR activation in a biochemically defined system. Conclusion: ATR signaling requires enlargement of the DNA excision gap by EXO1. Significance: The six excision repair factors, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of proteins for ATR-activation upon UV-induced DNA damage.

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“…4A). We and others have previously shown that TICRR loss causes a DNA damage checkpoint defect, so we wanted to test whether the failure to see increased H2AX phosphorylation was due to TICRR TESE interfering with the checkpoint response (Sansam et al 2010;Hassan et al 2013). Exposure of U2OS cells with or without TICRR TESE to ionizing radiation (IR) to cause dsDNA breaks resulted in a similar increase in gH2AX foci, indicating that TICRR TESE does not interfere with H2AX phosphorylation in response to DNA damage (Fig.…”

Section: Ticrr Tese Does Not Cause Dna Damagementioning

confidence: 99%

Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation

et al. 2015

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S-phase cyclin-dependent kinases (CDKs) stimulate replication initiation and accelerate progression through the replication timing program, but it is unknown which CDK substrates are responsible for these effects. CDK phosphorylation of the replication factor TICRR (TopBP1-interacting checkpoint and replication regulator)/ TRESLIN is required for DNA replication. We show here that phosphorylated TICRR is limiting for S-phase progression. Overexpression of a TICRR mutant with phosphomimetic mutations at two key CDK-phosphorylated residues (TICRR TESE ) stimulates DNA synthesis and shortens S phase by increasing replication initiation. This effect requires the TICRR region that is necessary for its interaction with MDM two-binding protein. Expression of TICRRTESE does not grossly alter the spatial organization of replication forks in the nucleus but does increase replication clusters and the number of replication forks within each cluster. In contrast to CDK hyperactivation, the acceleration of S-phase progression by TICRR TESE does not induce DNA damage. These results show that CDK can stimulate initiation and compress the replication timing program by phosphorylating a single protein, suggesting a simple mechanism by which S-phase length is controlled.

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Direct Role for the Replication Protein Treslin (Ticrr) in the ATR Kinase-mediated Checkpoint Response

Cited by 16 publications

References 31 publications

RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling

RHINO forms a stoichiometric complex with the 9-1-1 checkpoint clamp and mediates ATR-Chk1 signaling

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation

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