Post-translational activation of non-homologous DNA end-joining in Xenopus oocyte extracts

Said Aoufouchi, Tina Patrick, Howard D. Lindsay, Sydney Shall, Chris C. Ford

Résultats de recherche: Contribution à un journalArticleRevue par des pairs

2 Citations (Scopus)

Résumé

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 cdc2/cyclin B, or by incubation with purified cdc2/cyclin B. Dephosphorylation of egg proteins by alkaline phosphatase inhibited the ability to join non-homologous DNA ends. 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 μM 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.

langue originaleAnglais
Pages (de - à)518-525
Nombre de pages8
journalEuropean Journal of Biochemistry
Volume247
Numéro de publication2
Les DOIs
étatPublié - 1 janv. 1997
Modification externeOui

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