Site-specific Initiation of DNA Replication in Metazoan Chromosomes and the Role of Nuclear Organization (original) (raw)
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Replication of purified DNA in Xenopus egg extract is dependent on nuclear assembly
Journal of cell science, 1990
Purified DNA undergoes a single round of semiconservative replication when incubated in extracts of Xenopus eggs. These extracts also assemble purified DNA into pseudo-nuclei, structures closely resembling normal interphase nuclei. In this paper we show that although less than 60% of purified DNA is assembled into pseudo-nuclei, DNA replication takes place only within these pseudo-nuclei. Further, when nuclear assembly is prevented, the initiation of replication on purified DNA molecules does not occur. In contrast to previous reports, we show that the initiation of DNA replication occurs only during interphase and not during mitosis, even when very high concentrations of purified DNA are used. These experiments show that nuclear formation is a general requirement for the initiation of DNA replication in this system.
Selective activation of pre-replication complexes in vitro at specific sites in mammalian nuclei
Journal of Cell Science, 2000
As the first step in determining whether or not pre-replication complexes are assembled at specific sites along mammalian chromosomes, nuclei from G(1)-phase hamster cells were incubated briefly in Xenopus egg extract in order to initiate DNA replication. Most of the nascent DNA consisted of RNA-primed DNA chains 0.5 to 2 kb in length, and its origins in the DHFR gene region were mapped using both the early labeled fragment assay and the nascent strand abundance assay. The results revealed three important features of mammalian replication origins. First, Xenopus egg extract can selectively activate the same origins of bi-directional replication (e.g. ori-beta) and (beta') that are used by hamster cells in vivo. Previous reports of a broad peak of nascent DNA centered at ori-(beta/(beta)' appeared to result from the use of aphidicolin to synchronize nuclei and from prolonged exposure of nuclei to egg extracts. Second, these sites were not present until late G(1)-phase of the ...
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
Replication initiation complex formation in the absence of nuclear function in Xenopus
Nucleic acids research, 2009
In this article, we study how intercalation-induced changes in chromatin and DNA topology affect chromosomal DNA replication using Xenopus egg extracts. Unexpectedly, intercalation by ethidium or doxorubicin prevents formation of a functional nucleus: although nucleosome formation occurs, DNA decondensation is arrested, membranous vesicles accumulate around DNA but do not fuse to form a nuclear membrane, active transport is abolished and lamins are found on chromatin, but do not assemble into a lamina. DNA replication is inhibited at the stage of initiation complex activation, as shown by molecular combing of DNA and by the absence of checkpoint activation. Replication of single-stranded DNA is not prevented. Surprisingly, in spite of the absence of nuclear function, DNA-replication proteins of pre-replication and initiation complexes are loaded onto chromatin. This is a general phenomenon as initiation complexes could also be seen without ethidium in membrane-depleted extracts whic...
Nuclear structure and the control of DNA replication in the Xenopus embryo
Journal of Cell Science, 1989
Summary We have developed a cell-free system from frog eggs that efficiently initiates and completes a single round of semi-conservative replication. 70–100 % of sperm chromatin and up to 40 % of plasmid DNA molecules are completely replicated in vitro. Before DNA is replicated it is assembled into nuclei surrounded by a double unit membrane studded with nuclear pores. Flow cytometry shows that initiation events are co-ordinated within individual nuclei, although different nuclei can start to replicate at different times in the same extract. This demonstrates the importance of nuclear structure in the control of DNA replication in this system. Only a single round of semi-conservative replication occurs in the cell-free system. This mirrors the way that only one round of DNA replication occurs in each cell cycle in vivo. When replicated nuclei are transferred to fresh extract they are unable to undergo another round of replication. However, if the nuclear envelope is permeabilised be...
Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs
The Journal of Cell Biology, 1988
We have studied the pathway of nuclear assembly from demembranated sperm chromatin by fractionating a cell-free system from Xenopus eggs (Lohka, M. J., and Y. Masui. 1983. Science (Wash. DC). 220:719-721). Both the soluble fraction and a washed vesicular fraction are required for formation of normal nuclei that initiate replication in vitro. The soluble fraction alone decondenses chromatin and the vesicular fraction alone surrounds chromatin with membranes. Both fractions are required for formation of nuclear pore complexes. Recombining these two fractions recovers approximately 100% of the nuclear assembly and DNA replication activities. Restricting the proportion of the vesicular fraction slows acquisition of the nuclear membrane and allows observation of immature nuclear pores ("prepores"). These form as arrays around and within the chromatin mass before membranes form. Subsequently membrane vesicles bind to these prepores, linking them by a single membrane throughout t...
Dna Replication and Chromatin Assembly Using Xenopus Eggs or Embryos
1999
The investigation of the cellular and biochemical processes that are involved in eukaryotic chromosome replication has for many years lagged behind that of other biosynthetic processes such as transcription or translation. Except in yeast, small DNA molecules transfected in eukaryotic cells replicate very poorly and a genetic approach to DNA replication is difficult. The development of cell-free systems that can sustain the replication of viral DNA molecules was the first significant step towards the dissection of DNA replication at the molecular level . A fundamental stage of DNA replication, such as initiation, could not be satisfactorily analyzed, however because viral DNA replication is entirely dependent on a virus encoded protein. The development of cell-free systems from Xenopus eggs has provided the first non-viral cell-free systems capable of faithfully reproducing nuclear synthesis events, from the replication of the DNA to the assembly of chromatin and reconstitution of a functional nucleus. The large size of the egg (1.2 mm) permits the analysis of chromosomal replication in an in vivo context and relatively easy microinjection.
Journal of cell science, 1992
The lectin, wheat germ agglutinin (WGA), has previously been shown to prevent transport into the cell nucleus. This paper shows that WGA also inhibits nuclear DNA replication, under the same conditions that prevent transport. Although WGA eliminates sperm nuclear DNA replication in a cell-free extract of Xenopus eggs, DNA synthesis on a single-stranded template proceeds normally. Inhibition of nuclear DNA replication is partially reversed by addition of N-acetylglucosamine, and completely reversed by triacetylchitotriose. Sensitivity to inhibition by WGA is greatest during the nuclear assembly phase, and nuclear formation on sperm chromatin is blocked. DNA replication in preformed nuclear templates is also sensitive to WGA inhibition. I propose that WGA blocks DNA replication by preventing nuclear transport. The data presented here also indicate that, under certain circumstances, the elongation stage of DNA replication does not proceed in the absence of an intact nuclear envelope. T...
The Journal of cell …, 1996
Xenopus egg extracts initiate DNA replica-1. Abbreviations used in this paper. DAPI, 4',6-diamidino-2-phenylindole; DHFR, dihydrofolate reductase; 6-DMAP, 6-dimethylaminopurine; ELFH, early labeled fragment hybridization; MAR, matrix attachment region; OBR, origin of bidirectional replication; ODP, origin decision point; pre-CX, precondensation chromosome; pre-RC, prereplication complex; SAR, scaffold attachment region.
The EMBO journal, 1987
We have used a novel approach to investigate the control of initiation of replication of sperm nuclei in a Xenopus cell-free extract. Nascent DNA was labelled with biotin by supplementing the extract with biotin-11-dUTP, and isolated nuclei were then probed with fluorescein-conjugated streptavidin. Flow cytometry was used to measure the biotin content of individual nuclei and their total DNA content. This showed that incorporation of the biotinylated precursor increases linearly with DNA content. Haploid sperm nuclei replicate fully to reach the diploid DNA content over 2-6 h in the extract. Synthesis stops once the diploid DNA content is reached. Different nuclei enter S phase at different times over greater than 1.5 h, although they share the same cytoplasmic environment. Nuclei reach their maximum rates of synthesis soon after entry into S phase and some replicate fully in less than 0.5 h, resembling the rates of replication observed in the intact egg. These results indicate that...