Post‐Translational Activation of Non‐Homologous DNA End‐Joining in Xenopus Oocyte Extracts

Abstract

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...

The Mre11/Rad50/Nbs1 complex functions in resection-based DNA end joining in Xenopus laevis

“…In order to directly investigate a requirement for the MRN complex in NHEJ, we used a plasmid-based NHEJ assay based on that of Aoufouchi et al (63) to determine NHEJ efficiency in X. laevis extract immunodepleted for XMre11 or XNbs1. The NHEJ substrate was produced by digesting pUC19 plasmid with PstI and SmaI generating a linear DNA with blunt and 3′-overhang ends.…”

“…In order to investigate any requirement for the MRN complex in NHEJ in X. laevis , we initially conducted a plasmid-based NHEJ assay (63) using X. laevis egg extracts immunodepleted for XMre11 or XNbs1. Southern blot analysis of the end joining products indicated that NHEJ is unaffected in extracts lacking XMRN activity.…”

“…Twenty microlitres of egg extract was combined with 12.5 ng of linear DNA template and 1 µl of NHEJ mix (1 mM ATP, 1 mM MgCl 2 and 50 μM dNTPs) (63) and incubated at 21°C. Samples were then processed for analysis by agarose or acrylamide gel electrophoresis.…”

Nonhomologous DNA end joining in cell‐free systems

“…Unlike the crude extract, this partially purified DNA ligase (PPL) could join ends with cohesive PSSs but not mismatched ends. When the Xenopus egg extract was passed through a Sephacryl S300 gel‐filtration column in 0.1 m KCl, a pool of fractions (> 66 kDa) was recovered that supported NHEJ between nonmatching ends ( Pst I‐ Sma I) with an activity essentially as efficient and precise as in the crude extract [17]. A more extensive effort to fractionate the Xenopus egg extract was recently reported [20].…”

“…Poly(ADP‐ribose)polymerase (PARP) is another enzyme that is activated by DNA strand breaks (single or double stranded), but PARP knock‐out mice are not hypersensitive to DNA damage, suggesting that it is not directly involved in the DNA repair process (reviewed in [58]). Nevertheless, NHEJ in a partially purified fraction from the Xenopus egg extract (‘fraction Y’), but not in the crude extract, was found to be inhibited in the presence of the PARP‐inhibitor 3‐aminobenzamide [17]. Both the joining of nonmatching and cohesive ends was affected.…”