Can someone explain to me /give reference for how exactly does the Thymidine synchronization activate cell cycle checkpoint?
I know that it somehow decreases dCTP pools. Does anyone know exact mechanism?
Hi Rasika,
You may find your answer in the attached paper.
"To trap HeLa cells in S phase, inhibitors of DNA synthesis including thymidine, aphidicolin, and hydroxyurea are frequently used. High concentration of thymidine interrupts the deoxynu- cleotide metabolism pathway, thereby halting DNA replication. As treatment with thymidine arrests cells throughout S phase, a double thymidine block procedure (which involves releasing cells from a first thymidine block before trapping them with a second thymidine block) is generally used to induce a more synchronized early S phase blockade."
Guillaume
Dear Rasika
The paper sent by Guillaume is extremely good and succinctly describes the various methods for producing cell cycle arrest.
Was your question related to the methodology? Or did you want to know "Why should Thymidine produce S-Phase block and arrest since it is needed for DNA synthesis?'
Narayanan
Thanks Benjamin.
Yes. This can be called competitive inhibition. Increase the pool of compound A (damager) so that it blocks the binding sites of compound B which is necessary for proper metabolism. Just like giving Methotrexate so that cells cant utilize Folic acid.
Narayanan
I found the following paper helpful for understanding the mechanism behind double Thymidine block:
Gunnar Bjursell and Peter Reichard, 1973
http://www.jbc.org/content/248/11/3904.full.pdf
This protocol paper seems to be useful: Article Cell Synchronization by Double Thymidine Block
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