Aoufouchi, Said and Patrick, Tina and Lindsay, Howard D. and Shall, Sydney and Ford, Christopher C. (1997) Post-translational activation of non-homologous DNA end-joining in Xenopus oocyte extracts. FEBS Journal, 247 (2). pp. 518-525. ISSN 1742-464XFull text not available from this repository.
We have analysed the recircularisation of plasmid DNA, cut with two different endonucleases to generate non-homologous DNA ends, in extracts of unfertilised eggs and oocytes of Xenopus. We found that the capacity to join non-homologous DNA ends, generating diagnostic covalently closed monomer circles, appeared during oocyte maturation at the time of germinal vesicle breakdown. This enzyme function was post-translationally activated in oocyte extracts incubated with unfertilised egg extract containing active cdc2kyclin B, or by incubation with purified cdc2/cyclin B. Dephosphorylation of egg proteins by alkaline phosphatase inhibited the ability to join non-homologous DNA endr. We show that most linear non-homologous DNA ends repaired to form closed-circular supercoiled monomers, are joined without loss of nucleotides. Following partial purification, the activity was inhibited by inhibitors of poly(ADP-Rib) polymerase, an enzyme that is inactive in oocytes, but phosphorylated and activated during maturation. Competitive inhibition of poly(ADP-Rib) polymerase by > 50 pM 3-aminobenzamide prevented the joining of both matched and non-homologous DNA ends. We conclude that post-translational phosphorylation provides one route by which end-joining of non-homologous DNA can be regulated.
|Journal or Publication Title:||FEBS Journal|
|Uncontrolled Keywords:||non-homologous recombination ; Xenopus extracts ; poly(ADP-ribose) polymerase ; cyclindependent kinase ; post-translational control.|
|Subjects:||Q Science > QH Natural history > QH301 Biology|
|Departments:||Faculty of Health and Medicine > Medicine|
|Deposited By:||Dr Howard Lindsay|
|Deposited On:||17 Nov 2009 13:07|
|Last Modified:||26 Jul 2012 16:46|
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