A new in vitro system for activating the cell cycle checkpoint.
In response to DNA damage, cells launch elegant networks of genome surveillance mechanisms, called cell cycle checkpoints, to detect and repair damaged DNA to maintain the genome stability. Key components of cell cycle checkpoints are two PI3K-related protein kinases (PIKK), ATR and ATM, which participate in both sensing the DNA damage and transducing the damage signal through phosphorylating two target protein kinases, Chk1 and Chk2, respectively. However, how exactly cell cycle checkpoints are activated, maintained, and terminated are not completely understood. Given the complexity of the cell cycle checkpoint signaling and the cellular environment, systems that can faithfully mimic the cell cycle checkpoint activation in vitro, such as the Xenopus egg extracts, are of extreme value in dissecting the precise molecular mechanisms underlying DNA damage response. Here we describe that the well-established in vitro transcription and translation (IVTNT) system has the capability to induce protein phosphorylation of substrates for ATR or ATM, including Chk1, Rad17, and ATM itself. These phosphorylation events highly mimic those occurring in cells when treated with DNA damaging agents. Our results demonstrate that the IVTNT system could be developed into a novel in vitro system to facilitating the dissecting of mechanisms leading to cell cycle checkpoint activation in vivo.
Department of Pharmacology, Case Western Reserve University, Cleveland, OH, USA.
Jingna
J
Wang
Staci
S
Engle
Youwei
Y
Zhang
D064007:Ataxia Telangiectasia Mutated Proteins; D018929:Cell Culture Techniques; D002453:Cell Cycle / Q000502:physiology*; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology; D000071877:Checkpoint Kinase 1; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology; D042822:Genomic Instability; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology; D025521:Tumor Suppressor Proteins / Q000378:metabolism / Q000502:physiology; D029867:Xenopus Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C113575:Rad17 protein, human; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; D011494:Protein Kinases; C576324:ATM protein, human; 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
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