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.
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.
Guo
Z
Z
Division of Biology, 216-76, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
Kumagai
A
A
Wang
S X
SX
Dunphy
W G
WG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000502:physiology*; D002457:Cell Extracts; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004261:DNA Replication / Q000528:radiation effects; D018076:DNA, Complementary / Q000235:genetics; D004527:Egg Proteins / Q000502:physiology*; D005816:Genetic Complementation Test; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014466:Ultraviolet Rays / Q000009:adverse effects*; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000235:genetics / Q000502:physiology*
D018797:Cell Cycle Proteins; D002457:Cell Extracts; D018076:DNA, Complementary; D004527:Egg Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D004247:DNA; D011494:Protein Kinases; C576898:Atr protein, Xenopus; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases