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Study of the DNA Damage Checkpoint using Xenopus Egg Extracts
Published on: November 5, 2012
Chk1- and claspin-dependent but ATR/ATM- and Rad17-independent DNA replication checkpoint response in HeLa cells
Verónica Rodríguez-Bravo1, Sandra Guaita-Esteruelas, Roger Florensa
1Departament de Biologia Cellular i Anatomia Patològica, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain.
Abstract:
When DNA synthesis is inhibited, DNA replication checkpoint is activated to prevent mitosis entry without fully replicated DNA. In Xenopus, caffeine-sensitive kinases [ataxia telangiectasia mutated (ATM) and ATM-related protein (ATR)] are essential in this checkpoint response, but in mammal cells an ATR/ATM-independent checkpoint response to DNA synthesis inhibition exists. Using HeLa cells, which have a caffeine-insensitive checkpoint response, we have analyzed here which molecules known to be involved in the DNA replication checkpoint participate in the caffeine-insensitive response. When DNA synthesis was inhibited in the presence of UCN01 or after knocking down Chk1 expression [Chk1 small interfering RNA (siRNA)], HeLa cells entered into aberrant mitosis. Consequently, Chk1 is essential for both the ATR/ATM-dependent and ATR/ATM-independent checkpoint response in HeLa cells. Neither wortmannin, Ly294002, nor SB202190 abrogated the caffeine-insensitive checkpoint response, indicating that DNA-PK and p38 alpha,beta are not involved in the ATR/ATM-independent Chk1 activation upon DNA synthesis inhibition. Using siRNA to knock down Rad17 and claspin, two molecules involved in sensing stalled replication forks, we also showed that claspin but not Rad17 is essential for the ATR/ATM-independent checkpoint response. Inhibition of DNA synthesis in HeLa cells led to a decrease in cyclin B1 protein accumulation that was abrogated when UCN01 was added or when claspin was knocked down. We conclude that upon DNA synthesis inhibition, Chk1 can be activated in a claspin-dependent manner independently of ATR and ATM, leading to cyclin B1 down-regulation and providing the cells of an additional mechanism to inhibit mitosis entry.
Insights
HeLa cells utilize a caffeine-insensitive DNA replication checkpoint involving Chk1 kinase and claspin, independent of ATR/ATM, to prevent mitosis entry and down-regulate cyclin B1.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- DNA replication checkpoints prevent cell division before DNA is fully replicated.
- While Xenopus checkpoints rely on caffeine-sensitive kinases (ATM/ATR), mammalian cells possess alternative pathways.
- HeLa cells exhibit a caffeine-insensitive response to DNA synthesis inhibition.
Purpose of the Study:
- To identify molecules involved in the caffeine-insensitive DNA replication checkpoint in HeLa cells.
- To elucidate the ATR/ATM-independent pathway for DNA synthesis inhibition response.
- To understand the role of Chk1, Rad17, and claspin in this specific checkpoint.
Main Methods:
- Utilized HeLa cells with a caffeine-insensitive checkpoint.
- Employed UCN01 treatment and Chk1 small interfering RNA (siRNA) to inhibit DNA synthesis and Chk1.
- Investigated the involvement of DNA-PK, p38, Rad17, and claspin using specific inhibitors and siRNA knockdown.
- Monitored cyclin B1 protein levels.
Main Results:
- Chk1 kinase is essential for both ATR/ATM-dependent and independent checkpoint responses in HeLa cells.
- The caffeine-insensitive response was not affected by inhibitors of DNA-PK and p38.
- Claspin, but not Rad17, is crucial for the ATR/ATM-independent checkpoint activation.
- DNA synthesis inhibition led to decreased cyclin B1, which was rescued by UCN01 or claspin knockdown.
Conclusions:
- HeLa cells possess an ATR/ATM-independent DNA replication checkpoint pathway.
- This pathway involves claspin-dependent activation of Chk1, leading to cyclin B1 down-regulation.
- This provides an additional mechanism for inhibiting mitosis entry upon DNA synthesis inhibition.
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