Activation of a system for the joining of nonhomologous DNA ends during Xenopus egg maturation.

Goedecke, Wolfgang; Vielmetter, Walter; Pfeiffer, Petra

doi:10.1128/mcb.12.2.811

Cited by 35 publications

(27 citation statements)

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“…It remains to be seen whether this so-called alignment factor(s) is related to the apparatus that performs site-directed recombination of immunoglobulin and T-cell receptor genes (reviewed in Jackson and Jeggo, 1995;Weaver, 1995). The strong DNA-end joining activity is characteristic for frog eggs and early embryos; it was hardly, if at all, detectable in oocytes, and there is a dramatic boost of the activity when oocytes mature into unfertilized eggs (Goedecke et al, 1992). From studies by D. Carroll and colleagues (Carroll, 1983;Carroll et al, 1986) it is also known that Xenopus oocytes, like most cells studied so far, are able to join two overlapping identical sequences via homologous recombination.…”

Section: Discussion Homologous Recombination and Nonhomologous Dna-enmentioning

confidence: 99%

“…Furthermore, the reaction was predominantly intramolecular resulting exclusively in monomeric recircularized plasmids as revealed by isolation of plasmid DMA from Kan r -colonies (data not shown). Intramolecular joining is strongly favoured by injecting low amounts of DMA below the nanogram range (Pfeiffer and Vielmetter, 1988;Goedecke et a/., 1992) as used in this study.…”

Section: Fertilized Eggs Almost Exclusively Show Dma-end Joining Whimentioning

confidence: 99%

“…the number of Tef-colonies is proportional to the amount of injected DNA. However, there was little, if any, DNA-end joining activity in the oocytes even with very high amounts of injected DNA (Figure 4A-C; see also Goedecke et a/., 1992). Thus the DNA-end joining activity becomes the predominant recombination/repair pathway only after complete egg maturation whereas the homologous recombination activity remains constant throughout late germ cell maturation and embryonic development (Lehman efa/., 1993).…”

Section: Increasing Amounts Of Injected Dna Favours Homologous Recombmentioning

confidence: 99%

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Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

Hagmann¹,

Adlkofer²,

Pfeiffer³

et al. 1996

Biological Chemistry Hoppe-Seyler

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We have developed a versatile plasmid vector (pReco80% of the homologously recombined product, whereas in eggs a highly efficient DMA-end joining activity predominates Both reactions, homologous recombination and DMA-end joining, are shown to occur quickly, with the majority of the respective products being formed within the first 20 minutes of incubation under optimal conditions. In fertilized eggs, up to 50% of all injected linear DMA molecules are recircularized by DMA-end joining. With high amounts of injected DMA per fertilized egg, DMA-end joining is reduced, presumably due to competition for essential factors, and homologous recombination becomes readily detectable.As there is a sequence of rapid cleavage divisions after fertilization of the egg, the fast and highly efficient DMA-end joining, even though it is error-prone at the junction site, seems to be best suited to cope 1 present address: Institut für Zellbiologie, Swiss Federal Institute of Technology, Zürich-Hönggerberg, CH-8093 Zürich, Switzerland with DMA double-strand breaks that might occur in the genome during early embryogenesis. On the other hand, the long-lived oocytes seem to repair DMA double-strand breaks via homologous recombination. This latter property may be exploited both in Xenopus and in other organisms to achieve homologous integration of exogenous DMA into germ cells for gene targeting.

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Section: Discussion Homologous Recombination and Nonhomologous Dna-enmentioning

confidence: 99%

Section: Fertilized Eggs Almost Exclusively Show Dma-end Joining Whimentioning

confidence: 99%

Section: Increasing Amounts Of Injected Dna Favours Homologous Recombmentioning

confidence: 99%

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Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

Hagmann¹,

Adlkofer²,

Pfeiffer³

et al. 1996

Biological Chemistry Hoppe-Seyler

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“…However, following oocyte maturation, both simple ligation and non-conservative homologous recombination activities are detected in eggs and extracts of them, as is a third repair activity which joins DNA with non-homologous restriction termini [2,4,10, 111. This non-homologous end joining (NHEJ) activity will join any combination of restriction-digest ends of linear DNA presented as substrate, generating, at low DNA concentrations, diagnostic covalently closed monomer molecules [2, 71.…”

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confidence: 99%

“…Substrate DNA linearised with XhoI is ligated in early oocytes (stages I, 11 and III), but this activity is not observed in full grown oocytes (stageVI) even though ligation is part of the non-conservative homologous recombination activity and DNA ligase I and DNA ligase I1 activities are detected in stage-VI oocyte extracts [lo, 12, 131. A fivefold increase in exonuclease activity between stages I1 and VI oocytes may contribute to the failure to detect simple ligation products later in oogenesis [lo].However, following oocyte maturation, both simple ligation and non-conservative homologous recombination activities are detected in eggs and extracts of them, as is a third repair activity which joins DNA with non-homologous restriction termini [2,4,10, 111. This non-homologous end joining (NHEJ) activity will join any combination of restriction-digest ends of linear DNA presented as substrate, generating, at low DNA concentrations, diagnostic covalently closed monomer molecules [2, 71.…”

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

Aoufouchi

Patrick

Lindsay

et al. 1997

European Journal of Biochemistry

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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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Intermediates in extrachromosomal homologous recombination in Xenopus laevis oocytes: characterization by electron microscopy.

1993

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Several molecular mechanisms have been proposed to account for nonconservative homologous recombination. This type of recombination is particularly efficient in Xenopus oocytes when appropriate DNA substrates are injected. To distinguish between possible models, we have investigated recombination intermediates from oocytes by direct observation in the electron microscope. Partially recombined DNA was crosslinked with a psoralen derivative after incubation in oocytes to limit the branch migration that might occur during recovery procedures and alter the structures that were initially present. Branched structures, which we interpret as intermediates, represented approximately 10% of the DNA recovered and were readily analyzed. We did not observe any structures with internal loops predicted by invasion mechanisms. The majority of intermediates had one or two single‐stranded branches on product‐sized molecules, as predicted for incomplete junctions in the resection‐annealing mechanism. Detailed length measurements confirmed the expectations of that model. When recovered DNA was not crosslinked, or when annealed junctions were prepared in vitro, we saw branched structures that indicated the occurrence of extensive branch migration. Comparison with the crosslinked sample confirmed the effectiveness of the crosslinking in preserving structures created in the oocytes. Our results strongly support a resection‐annealing mechanism of recombination in oocytes.

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Activation of a system for the joining of nonhomologous DNA ends during Xenopus egg maturation.

Abstract: (21,34,36,38

Cited by 35 publications

References 50 publications

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

Dramatic Changes in the Ratio of Homologous Recombination to Nonhomologous DNA-End Joining in Oocytes and Early Embryos ofXenopus laevis

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

Intermediates in extrachromosomal homologous recombination in Xenopus laevis oocytes: characterization by electron microscopy.

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