Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts

Guo, Zijian; Kumagai, Akiko; Wang, Sophie X.; Dunphy, William G.

doi:10.1101/gad.842500

PreprintCited by 417DNA Repair MechanismsCancer-related Molecular PathwaysMicrotubule and mitosis dynamics

Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts

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Guo, Zijian, Kumagai, Akiko, Wang, Sophie X., Dunphy, William G.·2000·Genes & Development

DOI 10.1101/gad.842500PMC PMC317027

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Abstract

The checkpoint kinase Xchk1 becomes phosphorylated in Xenopus egg extracts in response to DNA replication blocks or UV-damaged DNA. Xchk1 is also required for the cell cycle delay that is induced by unreplicated or UV-damaged DNA. In this report, we have removed the Xenopus homolog of ATR (Xatr) from egg extracts by immunodepletion. In Xatr-depleted extracts, the checkpoint-associated phosphorylation of Xchk1 is abolished, and the cell cycle delay induced by replication blocks is strongly compromised. Xatr from egg extracts phosphorylated recombinant Xchk1 in vitro, but not a mutant form of Xchk1 (Xchk1-4AQ) containing nonphosphorylatable residues in its four conserved SQ/TQ motifs. Recombinant human ATR, but not a kinase-inactive mutant, phosphorylated the same sites in Xchk1. Furthermore, the Xchk1-4AQ mutant was found to be defective in mediating a checkpoint response in egg extracts. These findings suggest that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA.

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Chk1 is phosphorylated at serine 345 by ATR in response to stalled DNA replication forks and DNA damage induced by UV, ionizing radiation, methyl methanesulfonate, mitomycin C, and hydrogen peroxide.
“As a critical player in DDR, Chk1 is phosphorylated at Serine 345 by ATR in response to stalled DNA replication forks and DNA damage induced by UV, IR, methyl methanesulfonate (MMS), mitomycin C (MMC), and hydrogen peroxide [85–90].”
From Functional interplay between ATM/ATR-mediated DNA damage response and DNA repair pathways in oxidative stress (2014) · cites this paper as [85]

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Authors: Guo, Z., Kumagai, A., Wang, S. X., & Dunphy, W. G.
Affiliations: California Institute of Technology (US)
ORCID: Guo, Zijian
Kumagai, Akiko
Dunphy, William G.
Venue: Genes & Development
Publisher: Cold Spring Harbor Laboratory
Published: 2000-11-01
Volume: 14
Issue: 21
Pages: 2745-2756
ISSN: 0890-9369 (ISSN-L)
1549-5477
DOI: 10.1101/gad.842500
PMC: PMC317027
OpenAlex: W2112481873
Cited by: 417
References: 63
Citations by year: 2026: 6 · 2025: 3 · 2024: 4 · 2023: 8 · 2022: 6 · 2021: 8 · 2020: 10 · 2019: 10 · 2018: 12 · 2017: 10 · 2016: 15 · 2015: 10 · 2014: 8 · 2013: 13 · 2012: 16
Type: posted content
Language: en
Open access: bronze
doi.org
Locations: Genes & Development (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: Howard Hughes Medical Institute
National Institutes of Health
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Health Sciences › Medicine › Oncology › Cancer-related Molecular Pathways
Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
Keywords: ATR kinase, Chk1 phosphorylation, DNA replication checkpoint, UV-induced DNA damage, cell cycle arrest, Xenopus egg extracts, checkpoint kinase, DNA damage response
MeSH terms: Checkpoint Kinase 1, Amino Acid Sequence, Animals, Cell Cycle, Cell Extracts, DNA, DNA Damage, DNA Replication, Egg Proteins, Genetic Complementation Test, Humans, Models, Biological, Molecular Sequence Data, Oocytes, Phosphorylation, Protein Kinases, Protein Processing, Post-Translational, Recombinant Fusion Proteins, Species Specificity, Ultraviolet Rays, Xenopus laevis, Sequence Alignment, Protein Serine-Threonine Kinases, Sequence Homology, Amino Acid, DNA, Complementary, Cell Cycle Proteins, Amino Acid Substitution, Amino Acid Motifs, Xenopus Proteins, Ataxia Telangiectasia Mutated Proteins

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Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts

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