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.Keywords: non-homologous recombination; Xenopus extracts; poly(ADP-ribose) polymerase; cyclindependent kinase ; post-translational control.In all cells, maintenance of the integrity of the DNA is essential to survival. Thus, all cells have evolved sophisticated and multiple pathways which maintain the integrity of the DNA [l]. In some cells, DNA repair sometimes uses DNA recombination in the process of repair. In addition, during the production of both oocytes and spermatozoa, homologous recombination is an important process. This leads to the notion that the processes of DNA recombination might be controlled at several levels.It has been shown that, in oocytes and eggs of Xenopus luevis, various processes of DNA recombination can occur [2, 31. A slightly unusual example among these reactions is the rejoining of non-homologous DNA ends, which is evident in X. luevis eggs but not in oocytes [4]. This difference implies a dependance upon maturation of the oocyte and perhaps also on progression through the cell cycle.In Xenopus, meiotic recombination occurs in early stage-I oocytes before the frog is sexually mature [ 5 ] . Meiosis is suspended at diplotene while the oocyte grows, accumulating many components for use during embryogenesis (reviewed in [6] full grown (stage VI) oocyte resumes meiosis on hormonal stimulation and is shed as an unfertilised egg arrested in metaphase of meiosis 11. Several routes have been identified in Xenopus oocytes and eggs by which double-strand breaks can be closed 12, 3, 7-1 I]. In full grown oocytes, the dominant activity detected following injection of substrate DNA molecules into the oocyte nucleus (germinal vesicle) produces non-conservative homologous recombination through a resection-annealing proces...

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During the maturation of the oocytes of the frog Xenopus laevis, the rate of protein synthesis shows a twofold increase. Studies of the mechanisms involved in this stimulation have been seriously limited by the lack of an active cell-free translation system. We have now prepared such systems from oocytes, progesterone-matured oocytes and eggs of Xenopus laevis by induction of lysis by centrifugation of whole cells. The extracts are highly active in incorporation of labelled amino acids and, in the progesterone-matured and egg extracts, a substantial proportion of this is due to reinitiation on endogenous mRNA, as shown by the use of inhibitors. The increased rate of protein synthesis previously observed in intact oocytes following progesterone-induced maturation is reflected in the relative activities of the extracts. The difference in activity is not due to the presence of a dominant inhibitor of translation in the extracts from unstimulated oocytes. Labelling studies with initiator tRNA ([35S]Met-tRNAf) indicate a higher concentration of 43S preinitiation complexes in the extracts from unstimulated oocytes, suggesting an impairment of initiation of translation at or after the mRNA-binding step. Extracts from both oocytes and progesterone-matured oocytes translated endogenous mRNAs to give products ranging over a wide spectrum of molecular weight. However, significant translation of exogenous (globin) mRNA required the presence of reticulocyte postribosomal supernatant, suggesting that one or more factors required for mRNA recruitment is limiting in these extracts.

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