Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks.
ATR and Chk1 are protein kinases that perform major roles in the DNA replication checkpoint that delays entry into mitosis in response to DNA replication stress by hydroxyurea (HU) treatment. They are also activated by ionizing radiation (IR) that induces DNA double-strand breaks. Studies in human tissue culture and Xenopus egg extracts identified Claspin as a mediator that increased the activity of ATR toward Chk1. Because the in vivo functions of Claspin are not known, we generated Drosophila lines that each contained a mutated Claspin gene. Similar to the Drosophila mei-41/ATR and grp/Chk1 mutants, embryos of the Claspin mutant showed defects in checkpoint activation, which normally occurs in early embryogenesis in response to incomplete DNA replication. Additionally, Claspin mutant larvae were defective in G2 arrest after HU treatment; however, the defects were less severe than those of the mei-41/ATR and grp/Chk1 mutants. In contrast, IR-induced G2 arrest, which was severely defective in mei-41/ATR and grp/Chk1 mutants, occurred normally in the Claspin mutant. We also found that Claspin was phosphorylated in response to HU and IR treatment and a hyperphosphorylated form of Claspin was generated only after HU treatment in mei-41/ATR-dependent and tefu/ATM-independent way. In summary, our data suggest that Drosophila Claspin is required for the G2 arrest that is induced by DNA replication stress but not by DNA double-strand breaks, and this difference is probably due to distinct phosphorylation statuses.
Ilsong Institute of Life Science, Hallym University, Anyang, Republic of Korea.
Eun-Mi
EM
Lee
Tram Thi Bich
TT
Trinh
Hee Jin
HJ
Shim
Suk-Young
SY
Park
Trang Thi Thu
TT
Nguyen
Min-Joo
MJ
Kim
Young-Han
YH
Song
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000528:radiation effects; D053903:DNA Breaks, Double-Stranded; D004261:DNA Replication / Q000187:drug effects; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D059565:G2 Phase Cell Cycle Checkpoints; D005720:Gamma Rays; D006918:Hydroxyurea / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological; D029867:Xenopus Proteins
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D029867:Xenopus Proteins; C576616:claspin protein, Drosophila; D004247:DNA; D011494:Protein Kinases; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; C444435:Mei-41 protein, Drosophila; D017346:Protein Serine-Threonine Kinases; C514947:Tefu protein, Drosophila; C000605877:grp protein, Drosophila; D006918:Hydroxyurea