Post-translational activation of non-homologous DNA end-joining in Xenopus oocyte extracts.
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 microM 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.
Aoufouchi
S
S
MRC Laboratory of Molecular Biology, Cambridge, UK.
Patrick
T
T
Lindsay
H D
HD
Shall
S
S
Ford
C C
CC
D000818:Animals; D001483:Base Sequence; D001549:Benzamides / Q000494:pharmacology; D016203:CDC2 Protein Kinase / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005260:Female; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D009865:Oocytes / Q000502:physiology*; D010063:Ovum / Q000502:physiology; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013698:Templates, Genetic; D014020:Tissue Extracts / Q000378:metabolism; D014982:Xenopus laevis
D001549:Benzamides; D016213:Cyclins; D009838:Oligodeoxyribonucleotides; D014020:Tissue Extracts; C025160:3-aminobenzamide; D011065:Poly(ADP-ribose) Polymerases; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases; D011088:DNA Ligases; D000072481:DNA Ligase ATP