Homologous recombination catalyzed by a nuclear extract from Xenopus oocytes.

Lehman, Chris W.; Carroll, Dana

doi:10.1073/pnas.88.23.10840

Cited by 22 publications

(19 citation statements)

References 60 publications

(64 reference statements)

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“…Maryon and Carroll have similarly identified a 5'-3' exonuclease in Xenopus oocytes which acts on substrate DNA to generate recombination intermediates (29)(30)(31). In addition, nuclear extracts prepared from Xenopus oocytes have been shown to catalyze homologous recombination in vitro, using a mechanism consistent with SSA (22).…”

Section: Discussionmentioning

confidence: 99%

A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology.

Huang¹

1993

Mol. Cell. Biol.

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When two linear DNA molecules with overlapping, homologous ends were incubated with a yeast nuclear extract, they recombined at the region of homology to produce a joint molecule. We have identified a 5'-3' exonuclease in the extract that is likely to be responsible for the formation of the observed product. We propose that the exonuclease degrades each substrate to reveal regions of complementary sequence which anneal to form a recombinant product. Consistent with this model, we have partially purified the activity that promotes joint molecule formation and found it to cofractionate with a 5'-3' exonuclease activity through three consecutive chromatography steps. We have further characterized the reaction to determine the optimal length of homology. Substrates with homologous terminal overlaps of 29 to 958 bp were capable of product formation, whereas substrates with longer overlaps were not. Extracts prepared from a number of recombinationdefective or nuclease-deficient strains revealed no defect in exonuclease activity, indicating that the reaction is likely to be dependent upon the product of an as yet unidentified gene.The importance of strand-specific exonucleases in genetic recombination has been well documented. In prokaryotes, the recE gene product (exonuclease VIII), the T7 gene 6 product, and X exonuclease are all 5'-3' double-stranded exonucleases proven to play a role in recombination (reviewed in reference 41). Several models for recombination in eukaryotes also predict the involvement of exonucleases. In the double-strand break repair (DSBR) model, it is proposed that the DNA ends created at the break are degraded by a strand-specific exonuclease to create single-stranded tails which are able to invade homologous DNA, thus initiating the recombination process (51). In vivo studies have documented 5'-3' degradation of the broken ends formed at several double-strand breaks associated with recombination hot spots in the yeast Saccharomyces cerevisiae. After HO endonuclease cleavage at the AMT locus during mating-type switching, the DNA undergoes exonucleolytic degradation to produce a 3' tail on the distal side of the HO cut (55). This strand is believed to invade the intact donor of mating-locus information, thus initiating transfer of information. A double-strand break induced at theARG4 locus during meiosis is processed to leave a 3' single-stranded tail (48). This site has been shown to be a recombination initiation site (34), indicating that a 3' tail may be involved in the initiation of meiotic recombination. Similarly, a meiotic recombination hot spot created by the insertion of the LEU2 gene at the HIS4 locus is associated with a meiosis-specific doublestrand break that is processed to generate 3' single-stranded tails (5, 7).An exonuclease is also envisioned to act in the singlestrand annealing (SSA) model of recombination, which was proposed to explain certain recombination events in mammalian systems (24)(25)(26). In this model, strand-specific degradation by an exonuclease is predicted to...

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Section: Discussionmentioning

confidence: 99%

A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology.

Huang¹

1993

Mol. Cell. Biol.

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“…This resection may be necessary for strand exchange to be initiated in homologous recombination, but results of others (3a) also suggest that the 3'-to-5' activity of HPP1 may be required for end joining. Recent evidence from reactions involving extracts of both Xenopus oocytes and yeast nuclei also shows that exonuclease digestion is an important component in the homologous recombination of linear DNA molecules with overlapping homology (10,15). Alternatively, it is not implausible that the opening up of the termini to allow single-strand annealing between the partners might be accomplished by helicases.…”

Section: Discussionmentioning

confidence: 99%

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

Nicolás¹,

Young²

1994

Mol. Cell. Biol.

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Mammalian cells have a marked capacity to repair double-strand breaks in DNA, but the molecular and biochemical mechanisms underlying this process are largely unknown. A previous report has described an activity from mammalian cell nuclei that is capable of multimerizing blunt-ended DNA substrates (R. Fishel, M.K. Derbyshire, S.P. Moore, and C.S.H. Young, Biochimie 73:257-267, 1991). In this report, we show that nuclear extracts from HeLa cells contain activities which preferentially join linear plasmid substrates in either a head-to-head or tail-to-tail configuration, that the joining reaction is covalent, and that the joining is accompanied by loss of sequence at the junction. Sequencing revealed that there was a loss of a uniform number of nucleotides from junctions formed from any one type of substrate. The loss was not determined by any simple site-specific mechanism, but the number of nucleotides lost was affected by the precise terminal sequence. There was no major effect on the efficiency or outcome of the joining reaction with substrates containing blunt ends or 3' or 5' protruding ends. Using a pair of plasmid molecules with distinguishable restriction enzyme sites, we also observed that blunt-ended DNA substrates could join with those containing protruding 3' ends. As with the junctions formed between molecules with identical ends, there was uniform loss of nucleotides. Taken together, the data are consistent with two models for the joining reaction in which molecules are aligned either throughout most of their length or by using small sequence homologies located toward their ends. Although either model can explain the preferential formation of head-to-head and tail-to-tail products, the latter predicts the precise lossof nucleotides observed. These activities are found in all cell lines examined so far and most likely represent an important repair activity of the mammalian cell.

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“…Thus the assay presented here might be more sensitive than previously used techniques (Goedecke etal., 1992). Interestingly, there was more junction variability in the plasmids recovered from oocytes when compared to the ones from eggs, as measured by the length of Xhol/Sallexcised fragments (Figure 6; see also Lehman and Carroll, 1991;Lehman et ai, 1994). This probably reflects a stronger exonucleolytic resection and/or weaker protection of DNA ends by 'alignment factor(s)' in oocytes versus eggs.…”

Section: Low But Bona Fide Dna-end Joining Already In Stage VI Oocytesmentioning

confidence: 99%

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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Homologous recombination catalyzed by a nuclear extract from Xenopus oocytes.

Cited by 22 publications

References 60 publications

A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology.

A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology.

Characterization of DNA end joining in a mammalian cell nuclear extract: junction formation is accompanied by nucleotide loss, which is limited and uniform but not site specific.

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

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