DNA replication in cell-free extracts from Xenopus eggs is prevented by disrupting nuclear envelope function.

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. The roles of the nuclear envelope and active nuclear transport in DNA replication are discussed.

[1]  C. Dingwall,et al.  The nuclear membrane. , 1992, Science.

[2]  L. Cox,et al.  DNA replication occurs at discrete sites in pseudonuclei assembled from purified DNA in vitro , 1991, Cell.

[3]  C. Hutchison,et al.  The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs. , 1991, Journal of cell science.

[4]  R. Laskey,et al.  The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts , 1991, The Journal of cell biology.

[5]  K. Wilson,et al.  A lamin-independent pathway for nuclear envelope assembly , 1990, The Journal of cell biology.

[6]  L. Cox,et al.  Extracts from eggs and oocytes of Xenopus laevis differ in their capacities for nuclear assembly and DNA replication. , 1990, Journal of cell science.

[7]  J. Blow,et al.  Replication of purified DNA in Xenopus egg extract is dependent on nuclear assembly. , 1990, Journal of cell science.

[8]  D. Forbes,et al.  Reconstitution of biochemically altered nuclear pores: Transport can be eliminated and restored , 1990, Cell.

[9]  J. White,et al.  Replication occurs at discrete foci spaced throughout nuclei replicating in vitro. , 1989, Journal of cell science.

[10]  C. Hutchison,et al.  Changes in the nuclear distribution of DNA polymerase alpha and PCNA/cyclin during the progress of the cell cycle, in a cell-free extract of Xenopus eggs. , 1989, Journal of cell science.

[11]  R Berezney,et al.  Mapping replicational sites in the eucaryotic cell nucleus , 1989, The Journal of cell biology.

[12]  W. G. Kelly,et al.  Glycosylation in the nucleus and cytoplasm. , 1989, Annual review of biochemistry.

[13]  M. Willingham,et al.  Nuclear protein import: specificity for transport across the nuclear pore. , 1988, Experimental cell research.

[14]  C. Hutchison,et al.  The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro. , 1988, Development.

[15]  B. Stillman,et al.  Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo , 1988, Molecular and Cellular Biology.

[16]  C. Dreyer,et al.  The function of the nuclear envelope in nuclear protein accumulation , 1988, The Journal of cell biology.

[17]  J. Blow,et al.  A role for the nuclear envelope in controlling DNA replication within the cell cycle , 1988, Nature.

[18]  D. Newmeyer,et al.  Nuclear import can be separated into distinct steps in vitro: Nuclear pore binding and translocation , 1988, Cell.

[19]  C. Feldherr,et al.  Translocation of RNA-coated gold particles through the nuclear pores of oocytes , 1988, The Journal of cell biology.

[20]  J. Blow,et al.  Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs , 1988, The Journal of cell biology.

[21]  L. Gerace,et al.  Functional organization of the nuclear envelope. , 1988, Annual review of cell biology.

[22]  M. Dabauvalle,et al.  Inhibition of nuclear accumulation of karyophilic proteins in living cells by microinjection of the lectin wheat germ agglutinin. , 1988, Experimental cell research.

[23]  M. Yamaizumi,et al.  Reversible inhibition of protein import into the nucleus by wheat germ agglutinin injected into cultured cells. , 1987, Experimental cell research.

[24]  C. Hutchison,et al.  Periodic DNA synthesis in cell‐free extracts of Xenopus eggs. , 1987, The EMBO journal.

[25]  J. Blow,et al.  Nuclei act as independent and integrated units of replication in a Xenopus cell‐free DNA replication system. , 1987, The EMBO journal.

[26]  D. Newmeyer,et al.  Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores , 1987, The Journal of cell biology.

[27]  J. Newport Nuclear reconstitution in vitro: Stages of assembly around protein-free DNA , 1987, Cell.

[28]  J. Blow,et al.  Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs , 1986, Cell.

[29]  D. Jackson,et al.  Replication occurs at a nucleoskeleton. , 1986, The EMBO journal.

[30]  F. Dean,et al.  In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[31]  B. Stillman,et al.  Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells , 1985, Molecular and cellular biology.

[32]  D. Jackson,et al.  A general method for preparing chromatin containing intact DNA. , 1985, The EMBO journal.

[33]  C. Feldherr,et al.  Movement of a karyophilic protein through the nuclear pores of oocytes , 1984, The Journal of cell biology.

[34]  M. Kirschner,et al.  Regulation of the cell cycle during early Xenopus development , 1984, Cell.

[35]  M. Lohka,et al.  Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs , 1984, The Journal of cell biology.

[36]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[37]  M. Lohka,et al.  Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. , 1983, Science.

[38]  R. Harland,et al.  DNA synthesis in a cell-free system from Xenopus eggs: Priming and elongation on single-stranded DNA in vitro , 1982, Cell.

[39]  B. Mittelman,et al.  On control. , 1979, Dental management.

[40]  J. Gurdon Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal. , 1976, Journal of embryology and experimental morphology.