Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation (original) (raw)
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Biochimie, 1990
Using Xenopus laevis oocytes and unfertilized eggs, we have developed a system which allows the study of DN A repair upon microinjection of pBR 322 DNA which has been previously modified in vitro by N-acetyl-aminofluorene, under controlled conditions. In unfertilized eggs, an efficient repair of pBR-18AAF DNA takes place, leading to a restoration of the transforming activity of the plasmid DNA towards Escherichia coil The repaired DNA is even efficiently replicated, the egg being "activated" by the microitljection. In the oocyte, a partial repair is observed as shown by the incorporation of labelled dCTP in the modified plasmid DNA, even in the presence of aphidicolin, an inhibitor of DNA polymerase or. However, the repair appears to be very limited, since it does not restore the transforming activity of the modified plasmid DNA. This inefficient repair in the oocyte may be due to the rapid packaging of foreign DNA into a minichromosome and / or to a very low level of DNA polymerase/~. This system was used to study the effect of diadenosine tetraphosphate (Ap~A) on DNA repair. Ap4A seems not to interfere with repair processes in the oocyte, but significantly inhibits the replication following the repair of AAF-modified plasmid DNA in unfertilized eggs. These results suggest that Ap4A could be involved in switching off the replication machinery when DNA is badly damaged. thus helping to avoid the perpetuation of DNA modifications in the daughter cells. This hypothesis is consistent with many previous reports on the accumulation of dinucleoside polyphosphates under stress conditions, which are known to result in modification of DNA.
Journal of Cell Science, 1990
We describe a cell-free extract derived from the oocytes of Xenopus laevis. The oocyte extract is capable of decondensing sperm chromatin and of replicating single-stranded DNA in a semiconservative, aphidicolin-sensitive manner. In addition, oocyte extract supports the elongation phase of DNA synthesis in nuclei that have been preinitiated for replication. All of these properties are shared by previously described egg extracts. However, oocyte extracts differ from egg extracts in two important ways. First, they cannot support nuclear assembly, as visualised by phase-contrast, fluorescence and electron microscopy. Second, they do not initiate replication on chromatin or nuclei de novo. Crude low-speed supernatants can be partially fractionated into soluble and vesicular components by high-speed centrifugation. Such fractions from eggs can be functionally reconstituted, but the oocyte soluble fraction does not acquire the ability to assemble nuclei, or replicate them, even when supplemented with the egg vesicular fraction. Similarly, oocyte vesicles cannot substitute for egg vesicles on reconstitution with the egg soluble fraction. When the requirement for nuclear assembly is bypassed by using preformed, quiescent nuclei, replication is observed in egg but not oocyte extracts. However, the oocyte extract is not inhibitory for initiation of replication, as it does not prevent replication of sperm nuclei when mixed with egg extract. We suggest that the different capabilities of egg and oocyte extracts could provide the basis of an assay system for identifying factors involved in the initiation of DNA replication.
Purification of a DNA-binding protein from Xenopus laevis unfertilized eggs
Nucleic Acids Research, 1977
A DNA-binding protein from Xenopus laevis unfertilized eggs has been purified to apparent homogeneity. It is a heat stable, lysine-rich protein and has a molecular weight corresponding to 8, 200 daltons, measured by sodium dodecyl sulphate gel electrophoresis. The protein, which is active in a monomeric form, stimulates DNA polymeraseo ,a, binds to single and double stranded DNA. One egg contains about 4 x 10 molecules (minimum estimate) of the protein; since we calculate that 4 x 10 molecules are sufficient to cover the entire genome (haploid complement), there is much more protein than is needed to cover chromosomal DNA.
Nucleotide Excision Repair in Oocyte Nuclear Extracts from Xenopus laevis
Methods, 2000
We have developed efficient DNA repair extracts derived from the unusually large nuclei of Xenopus oocytes. These extracts use nucleotide excision repair (NER) to completely remove bulky adducts from DNA. There is very little or no synthesis on control, undamaged DNA, indicating the extracts do not have significant nonspecific nuclease activity, and repair of cyclobutane pyrimidine dimers (CPDs) occurs in the dark, indicating that NER, and not photolyase, is responsible for CPD repair. The extracts can be inactivated with antibodies specific to repair proteins and then repair activity can be restored by adding purified recombinant protein. Here we describe detailed protocols for preparing Xenopus nuclear repair extracts.
Mitochondrial DNA polymerase from Xenopus laevis oocytes
Biochemical and Biophysical Research Communications, 1979
The DNA polymerase of Xenopus laevis oocytes, with characteristics similar to those of the y polymerases of other systems, can be extracted from the mitochondrial pellet and from the mitochondria purified on a sucrose gradient. It elutes from phosphocellulose at about 0.4 M KCI and sediments faster than 4 S in high salt glycerol gradients. At low KCI concentrations it uses Poly(A) better than activated DNA as a template; the reverse is true at KCI concentrations higher than 150 mM. Various experiments are presented that indicate that it is the mitochondrial DNA pol~n~erase. In a previous report we have described the major DNA polymerase activity able to use Poly(A)-oligo(dT) as a template in Xengpus laevis oocytes (l). This enzyme has characteristics similar to those of the ~ pol3anerases of other systems, in that it efficiently uses Poly(A).oligo(dT) as a template, has a sedimentation coefficient higher than 4S and is sensitive to N'ethylmaleimide (2). The enzyme has a strict cytoplasmic location in these cells and is present in a particulate structure so that detergent is needed for its solubilization (I, 3). When detergent was omitted during the homogenization of the oocytes, virtually all the enzyme could be extracted from a 12,000 xg pellet; since mitochondria are the major component of this pellet, we decided to investigate the possibility that the enzyme is the mitochondrial DNA polymerase. In this paper we present experiments that support this hypothesis. MATERIALS AND METHODS Materials. Deoxynucleotide-5'-triphosphates were obtained from Schwartz Mann, Orangeburg, N.Y.
Biochimica et biophysica acta (N), 1986
Diadenosine 5',5'"-Pt,p4-tetraphosphate (Ap4A) stimulates DNA synthesis in Xenopus iaevis oocytes in the presence of activated DNA as template. Besides Ap4A , other analogues such as AI~A, ATP and other derivatives are able to stimulate DNA polymerase activity. The effect of Ap4 A on DNA synthesis is observed with poly(dT) and poly(dT)-poly(dA) as templates, while no effect is found with poly(dA)(dT)12_18 and poly(dC)(dG)12_is. In the presence of a poly(dT) template, the oocyte extract is able to utilize Ap4A as primer and to form a covalent bond between this dinucleotide and the nascent poly(dA) chain. An Ap4A-binding protein present in the system has been purified and separated from DNA polymerase a-primase after phosphoceilulose chromatography. After this separation, Ap4 A is no longer able to stimulate the polymerase activity, or to be utilized as primer by DNA polymerase a-primase.
Initiation of DNA replication in xenopus egg extracts
Frontiers in Bioscience, 2004
Introduction 3. DNA replication during the early embryonic cell cycles 4. In vitro DNA replication: interphase, cycling, and mitotic Xenopus egg extracts 5. Indirect requirement for the nucleus in DNA replication 6. Sequence-independent replication initiation in Xenopus embryos 7. Pre-replication complex assembly: the helicase delivery mechanism 8. Pre-initiation complex assembly: the helicase activation step 9. A speculative model for helicase activation 10. Origin-recruitment of DNA polymerases 11. Regulation of Re-replication 12. The random completion problem 13. Conclusions and perspectives 14. Acknowledgements 15. References
Mismatch repair involving localized DNA synthesis in extracts of Xenopus eggs
Proceedings of the National Academy of Sciences, 1989
Repair of heteroduplex DNA containing G.T or A.C mismatches or containing two tandem unpaired bases occurred in vitro with Xenopus egg extracts as detected by a physical assay. The repair was accompanied by a mismatch-stimulated and mismatch-localized DNA synthesis. Repaired molecules, separated from unrepaired molecules, showed a 20- to 100-fold increase in DNA synthesis in the region of the mismatch compared to regions distant from the mismatch. The remaining unrepaired heteroduplex DNA included molecules that also displayed mismatch-stimulated DNA synthesis in the mismatch-proximal regions. These may represent intermediates in the repair process. The patterns of DNA synthesis suggest that repair begins at some distance from the mismatch and that as much as 1 kilobase or more can be involved in the mismatch-stimulated synthesis.
Proceedings of the National Academy of Sciences, 1996
In Xenopus egg extracts, DNA strand breaks (nicks) located 3' or 5' to a mismatch cause an overall 3-fold stimulation of the repair of the mismatch in circular heteroduplex DNA molecules. The increase in mismatch repair is almost entirely due to an increase in repair of the nicked strand, which is stimulated 5-fold. Repair synthesis is centered to the mismatch site, decreases symmetrically on both sides, and its position is not significantly altered by the presence of the nick. Therefore, it appears that in the Xenopus germ cells, the mismatch repair system utilizes nicks as signals for the induction and direction of mismatch repair, but not as the start or end point for excision and resynthesis.