Open AccessPost‐Translational Activation of Non‐Homologous DNA End‐Joining in Xenopus Oocyte Extracts
Author(s) -
Aoufouchi Said,
Patrick Tina,
Lindsay Howard D.,
Shall Sydney,
Ford Chris C.
Publication year - 1997
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1997.00518.x
Subject(s) - dna , xenopus , homologous chromosome , microbiology and biotechnology , germinal vesicle , biology , oocyte , dna polymerase , biochemistry , polymerase , dna clamp , polymerase chain reaction , embryo , gene , reverse transcriptase
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 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.