POSTTRANSLATIONAL ACTIVATION OF NONHOMOLOGOUS DNA END-JOINING IN XENOPUS OOCYTE EXTRACTS

We have analysed the recircularisation of plasmid DNA, cut with two di fferent endonuclenses to generate non-homologous DNA ends, in extracts of unfertilised eggs and oocytes of Xenopus. We found that the capaci ty to join non-homologous DNA ends, generating diagnostic covalently c losed monomer circles, appeared during oocyte maturation at the time o f germinal vesicle breakdown. This enzyme function was post-translatio nally activated in oocyte extracts incubated with unfertilised egg ext ract containing active cdc2/cyclin B, or by incubation with purified c dc2/cyelin B. Dephosphorylation of egg proteins by alkaline phosphatas e inhibited the ability to join non-homologous DNA ends. We show that most linear non-homologous DNA ends repaired to form closed-circular s upercoiled monomers, are joined without loss of nucleotides. Following partial purification, the activity was inhibited by inhibitors of pol y(ADP-Rib) polymerase, an enzyme that is inactive in oocytes, but phos phorylated and activated during maturation. Competitive inhibition of poly(ADP-Rib) polymerase by > 50 mu M 3-aminobenzamide prevented the j oining of both matched and non-homologous DNA ends. We conclude that p ost-translational phosphorylation provides one route by which end-join ing of non-homologous DNA can be regulated.