DNA-dependent phosphorylation of Chk1 and Claspin in a human cell-free system

Clarke, Catriona; Clarke, Paul R.

doi:10.1042/bj20041966

Cited by 86 publications

(97 citation statements)

References 44 publications

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“…Claspin increases the affinity of ATR for Chk1 (11). Mutations in the Chk1 binding domain of Claspin abrogate the phosphorylation of Chk1 by ATR (17,18,21,22). Here, we report that the same mutations also abrogate the ability of Claspin to function when tethered to DNA both in vitro and in vivo.…”

Section: Discussionmentioning

confidence: 52%

Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

Lindsey-Boltz

Sancar

2011

Journal of Biological Chemistry

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The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase II␤-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.DNA damage checkpoints delay cell cycle progression in response to DNA damage to maintain genomic integrity. In mammalian cells, checkpoint response signaling pathways are initiated primarily by two large phosphoinositide 3-kinase-related serine-threonine kinases, ataxia-telangiectasia mutated (ATM) 2 and ATM and RAD3-related (ATR). ATM is activated mainly, but not exclusively, by double-stranded breaks, and ATR is activated by ultraviolet (UV) and UV-mimetic chemical agents as well as other conditions that result in replication fork stalling. Upon activation, ATR activates the key signal-transducing kinase Chk1 by phosphorylating it on Ser 317 and Ser 345(1, 2), and the mediator protein Claspin is required for the efficient phosphorylation of Chk1 (3-5). ATR activation also requires topoisomerase II␤-binding protein 1 (TopBP1), which has been shown to activate ATR directly in defined systems in vitro (6 -11) as well as in vivo (6, 12). The current model for ATR activation is as follows. Singlestranded DNA (ssDNA) generated at sites of DNA damage during repair, transcription, or replication is bound by replication protein A (RPA), which then recruits ATR through a physical interaction between RPA and the ATR-binding partner, ATRinteracting protein (ATRIP). Independently, Rad17-RFC loads the 9-1-1 (Rad9-Rad1-Hus1) checkpoint complex at primer/ template junctions, where it recruits TopBP1 in the proximity of ATR. We have previously described an in vitro system that recapitulates ATR phosphorylation of Chk1 dependent on RPA-coated ssDNA and TopBP1 (8). However, RPA is not required for maximal ATR kinase activity in this system when the ssDNA is replaced with DNA containing bulky DNA base adducts (9, 10). In fact, we found that TopBP1 binds directly to damaged DNA and that the DNA binding activity of TopBP1 is required to confer damaged DNA-dependent activation of ATR. Thus, we hypothesized that TopBP1 may directly recognize damag...

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Section: Discussionmentioning

confidence: 52%

Tethering DNA Damage Checkpoint Mediator Proteins Topoisomerase IIβ-binding Protein 1 (TopBP1) and Claspin to DNA Activates Ataxia-Telangiectasia Mutated and RAD3-related (ATR) Phosphorylation of Checkpoint Kinase 1 (Chk1)

Lindsey-Boltz

Sancar

2011

Journal of Biological Chemistry

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“…Chapter (Clarke and Clarke, 2005) is phosphorylated even in the absence of replication stress (Wilsker et al, 2008). Both Ser345 and Ser317 are critical for CHK1 kinase function as serine-to-alanine substitution mutations (S345A or S317A) that eliminate phosphorylation at these sites leading to suppress checkpoint activation and reduce viability (Shimada et al, 2008;Walker et al, 2009;Wilsker et al, 2008).…”

Section: Ej 24hrmentioning

confidence: 99%

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors

2014

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“…Downregulation of human Claspin Lin et al, 2004) decreases Chk1 activation in response to hydroxyurea (HU), ionizing radiation (IR) and UV, while immunodepletion of Xenopus Claspin (Kumagai and Dunphy, 2000) inhibits Chk1 activation induced by DNA templates in egg extracts. In addition, it has been shown in vitro for both Xenopus (Kumagai et al, 2004) and human systems (Clarke and Clarke, 2005) that the presence of Claspin is required for the ATR/ATM-dependent phosphorylation of Chk1. Furthermore, Claspin has been shown to be involved in regulation of the S-phase checkpoint, cell survival, and prevention of premature mitosis Lin et al, 2004).…”

Section: Introductionmentioning

confidence: 99%