Joining of nonhomologous DNA double strand breaks<i>in vitro</i>

Pfeiffer, Petra; Vielmetter, Walter

doi:10.1093/nar/16.3.907

Cited by 162 publications

(159 citation statements)

References 44 publications

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“…already after 10 min of incubation ( Figure 2A). Thus these data are in good agreement with previous work showing a very efficient DMA-end joining activity in Xenopus eggs (Pfeiffer and Vielmetter, 1988;Thode et a/., 1990). 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).…”

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

confidence: 93%

“…In this study, the substrate DMA, together with the internal reference plasmid pCml 84, is microinjected into Xenopus laevis stage VI oocytes and fertilized eggs which are known to support homologous recombination and nonhomologous DMA-end joining, respectively (Carroll, 1983;Carroll etal., 1986;Pfeiffer and Vielmetter, 1988; see also Rusconi and Schaffner, 1981). We were able to extend current knowledge about recombination in these cells and show that, under optimal conditions, both events are very fast with the majority of the respective products being produced within the first 20 min after injection.…”

Section: Introductionmentioning

confidence: 56%

“…Fertilized eggs and early embryos of the African clawed frog Xenopus laevis are able to efficiently join any two DNA ends by a nonhomologous recombination mechanism that is referred to as DNA-end joining or end-to-end joining (Pfeiffer and Vielmetter, 1988; see also Rusconi and Schaffner, 1981). From detailed studies with model sub-strates, it is known that this DMA-end joining activity is more sophisticated than originally anticipated.…”

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%

“…As an alternative explanation, the seeming inhibition of DNA-end joining at high DNA concentrations might be due to concatemer formation instead of intramolecular recircularization. Such concatemers are known to transform bacteria very inefficiently (Pfeiffer and Vielmetter, 1988). This latter mechanism would however not explain why only DNA-end joining, but not homologous recombination, is adversely affected by increasing DNA concentrations.…”

Section: Competition For Limiting Factor(s) At High Dna Concentrations?mentioning

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: Fertilized Eggs Almost Exclusively Show Dma-end Joining Whisupporting

confidence: 93%

Section: Introductionmentioning

confidence: 56%

Section: Discussion Homologous Recombination and Nonhomologous Dna-enmentioning

confidence: 99%

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

confidence: 99%

Section: Competition For Limiting Factor(s) At High Dna Concentrations?mentioning

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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Sequence homologies, N sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation.

1990

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Sequence analysis of rearranged VHDJH genes of B lineage cells from various stages of ontogeny indicates that short sequence homologies at the breakpoints of recombination contribute to V region gene assembly. Such homologies are regularly seen at DJH junctions of neonatal pre‐B cells, most of which do not contain N sequences. In the same cells, but not at later developmental stages, preferential usage of the JH1 element is observed. After birth, N sequence insertion increases with time and is always more prominent at the VHD border than the DJH border. In pre‐B cells from adult animals and in mature B cells, in cases where N sequences were not detectable, sequence homologies at the DJH border were found in only half of the instances. This lower incidence could be due to N sequence addition to one of the recombining DNA ends and/or cellular selection. Inspection of VHDJH junctions for N sequence insertion, sequence homologies at the DJH border and JH1 usage allows the estimation of the timepoint in ontogeny at which particular B cell subsets are seeded into the immune system. Specifically, the present data show that the cells of the Ly1 B cell subset are generated not only neonatally but also beyond the first weeks of life. However, the DJH junctions of the progenitors of chronic B cell leukemias which originate from the same B cell subset resemble those of neonatal pre‐B cells, suggesting that these cells have already undergone a transforming event at this early developmental stage.

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Morphological transformation of 10T1/2 mouse embryo cells can be initiated by DNA double‐strand breaks alone

Borek

Ong

Morgan

et al. 1991

Molecular Carcinogenesis

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Malignant transformation of mouse fibroblasts was produced by electroporation with restriction enzymes. Similar transformation frequencies were observed with Pstl, Pvull, and Xbal, which cut genomic DNA at similar overall frequencies but have different termini, i.e., a 3' overhang, a blunt end, and a 5' overhang, respectively. The dose-response curve for restriction enzyme transformation shows a marked plateau in frequencies of transformed foci per surviving cell, whereas x-irradiation of the same cells gives a linear dose-response curve. Evidently, transformation can be caused by DNA double-strand breaks alone at a limited number of sites, but the evidence from x rays suggests that other kinds of DNA damage can cause transformation independently.

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Joining of nonhomologous DNA double strand breaksin vitro

Cited by 162 publications

References 44 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

Sequence homologies, N sequence insertion and JH gene utilization in VHDJH joining: implications for the joining mechanism and the ontogenetic timing of Ly1 B cell and B-CLL progenitor generation.

Morphological transformation of 10T1/2 mouse embryo cells can be initiated by DNA double‐strand breaks alone

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