<b>A novel nuclease activity from <i>Xenopus laevis</i> releases short oligomers from 5′‐ends of double‐and single‐stranded DNA</b>

Reichenberger, Susanne; Brüll, Nicole; Feldmann, Elke; Göttlich, Bernd; Vielmetter, Walter; Pfeiffer, Petra

doi:10.1046/j.1365-2443.1996.d01-245.x

Cited by 5 publications

(4 citation statements)

References 38 publications

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“…Obviously, free DMA ends are not just repaired by DMA polymerase and then ligated. It was concluded that the DNA ends are juxtaposed by some cellular factor (Thode et al, 1990;Pfeiffer et al, 1994a;Pfeiffer et al, 1994b;Reichenberger et al, 1996) because the repair polymerase can also copy across two unligatable single-strands. 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).…”

Section: Discussion Homologous Recombination and Nonhomologous Dna-enmentioning

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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Section: Discussion Homologous Recombination and Nonhomologous Dna-enmentioning

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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“…The protocol for the construction of hairpin substrates from a constant stem oligonucleothe fusion protein. pQXpolβ-transformed M15 cells were tide and a variable linker oligonucleotide is described in detail, grown to A 600 of 0.7Ϫ0.9 in 500 ml Luria-Bertani medium supelsewhere [37,38]. For incorporation of the [ 32 P]phosphate label plemented with 100 mg/l ampicillin and 25 mg/l kanamycin.…”

Section: Overexpression Of Xenpolβ In E Coli and Purification Ofmentioning

confidence: 99%

“…To investigate whether Xenopus Polβ alone was capable of performing NEJ-like reactions in vitro via discontinuous DNA synthesis, we subjected the purified enzyme to NEJ assays using synthetic hairpin-shaped oligonucleotides. These hairpins, which consisted of internally [ 32 P]phosphate-labelled double-stranded stem-loop structures bearing freely variable 5′-PSS or 3′-PSS that serve as DSB termini, had been employed previously to successfully explore the capacities and limitations of NEJ in the Xenopus system [37,38].…”

Section: Overexpression Of Xenpolβ In E Coli and Purification Ofmentioning

confidence: 99%

“…overlap products. This reaction is most efficient with short PSS tails (up to 9 nucleotides) but ceases completely at a PSS length of about 12Ϫ13 nucleotides [37,38]. By contrast, Xenopus Polβ DISCUSSION was not able to use a 2-bp or a 4-bp primer to initiate the formation of overlap products, suggesting that Xenopus Polβ alone In this study we have reported the cloning of a functional Polβ cDNA from X. laevis and preparation of large-scale quanti-cannot align two DNA molecules paired by 2 bp or 4 bp.…”

Section: Formation Of Fold-back Products By Polβmentioning

confidence: 99%

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Cloning, purification and characterization of DNA polymerase β from Xenopus laevis

Reichenberger

Pfeiffer

1998

European Journal of Biochemistry

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Double-strand breaks in the DNA of vertebrate cells are joined by mechanisms of non-homologous DNA-end joining (NEJ). In extracts from Xenopus eggs, NEJ is inhibited by dideoxynucleotides, indicating a possible involvement of DNA polymerase β (Polβ). Since some types of NEJ products were shown to be formed in vitro by prokaryotic DNA polymerases lacking exonuclease activity, we were interested in whether Polβ alone would be capable of catalyzing NEJ reactions. Therefore we have cloned the fulllength cDNA of the Xenopus laevis Polβ. The cDNA, predicting a highly conserved 39-kDa protein of 334 amino acids, was tagged with six histidine residues at its N-terminus for overexpression in Escherichia coli, purified to near homogeneity, and shown to have the same catalytic properties as the previously cloned rat and human enzymes. Using oligonucleotides as substrates we show that the recombinant Xenopus Polβ adds single untemplated nucleotides to blunt ends. However, under conditions that permit efficient NEJ in Xenopus egg extracts, Polβ does not form those types of NEJ products formed by the prokaryotic polymerases indicating that Polβ alone is not able to mediate the complex NEJ process in vitro. Using substrates with 3′ protruding single strands of increasing length (6Ϫ16 nucleotides) we show that Polβ initiates fill-in DNA synthesis on fold-back structures formed by the longest 3′ protruding stand. This unusual feature of β-type polymerases requires that the loop of the fold-back structure consists of at least six bases and the stem be paired by at least 2bp to facilitate priming of DNA synthesis.Keywords : DNA polymerase β; Xenopus laevis ; non-homologous DNA-end joining; discontinuous DNA synthesis; untemplated base addition.The repair of chromosome breaks is essential to cell survival. ways may differ with respect to accuracy (i.e. preservation versus loss of terminal DSB sequences) they are conserved from Double-strand breaks (DSB), potentially lethal lesions in chromosomal DNA, may arise spontaneously [1,2] or after exposure yeast to man [9Ϫ16].Using an in vitro joining system based on extracts from Xento DNA-damaging agents, such as ionizing radiation [3]. In vertebrate cells, the major pathway of DSB repair proceeds by ille-opus laevis eggs and linear plasmid substrates bearing defined restriction ends we have shown earlier that a highly accurate gitimate recombination, which is able to rejoin DSB termini directly by non-homologous DNA-end-joining (NEJ) [1,4]. Un-NEJ mechanism generates two major types (overlap and fill-in) of products [10]. The type of product formed depends on the like homologous recombination, NEJ can proceed in the absence of sequence homology provided by the homologue or sister structure of the ends being joined: overlap products typically arise during the joining of DNA ends containing anti-parallel chromatid. Thus, two initially physically unlinked DNA ends must be aligned in some form of synapsis to counteract diffu-protruding single strands (PSS). By pairing of single complementary...

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Mechanisms of Non-Homologous DNA End Joining:Aspects of In Vitro Assays

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**A novel nuclease activity from Xenopus laevis releases short oligomers from 5′‐ends of double‐and single‐stranded DNA**

Cited by 5 publications

References 38 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

Cloning, purification and characterization of DNA polymerase β from Xenopus laevis

Mechanisms of Non-Homologous DNA End Joining:Aspects of In Vitro Assays

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