Nuclear proteins of quiescent Xenopus laevis cells inhibit DNA replication in intact and permeabilized nuclei.
Quiescent cells from adult vertebrate liver and contact-inhibited or serum-deprived tissue cultures are active metabolically but do not carry out nuclear DNA replication and cell division. Replication of intact nuclei isolated from either quiescent Xenopus liver or cultured Xenopus A6 cells in quiescence was barely detectable in interphase extracts of Xenopus laevis eggs, although Xenopus sperm chromatin was replicated with approximately 100% efficiency in the same extracts. Permeabilization of nuclei from quiescent Xenopus liver or cultured Xenopus epithelial A6 cells did not facilitate efficient replication in egg extracts. Moreover, replication of Xenopus sperm chromatin in egg extracts was strongly inhibited by a soluble extract of isolated Xenopus liver nuclei; in contrast, complementary-strand synthesis on single-stranded DNA templates in egg extracts was not affected. Inhibition was specific to endogenous molecules localized preferentially in quiescent as opposed to proliferating cell nuclei, and was not due to suppression of cdk2 kinase activity. Extracts of Xenopus liver nuclei also inhibited growth of sperm nuclei formed in egg extracts. However, the rate and extent of decondensation of sperm chromatin in egg extracts were not affected. The formation of prereplication centers detected by anti-RP-A antibody was not affected by extracts of liver nuclei, but formation of active replication foci was blocked by the same extracts. Inhibition of DNA replication was alleviated when liver nuclear extracts were added to metaphase egg extracts before or immediately after Ca++ ion-induced transition to interphase. A plausible interpretation of our data is that endogenous inhibitors of DNA replication play an important role in establishing and maintaining a quiescent state in Xenopus cells, both in vivo and in cultured cells, perhaps by negatively regulating positive modulators of the replication machinery.
Fang
J
J
Department of Zoology and Genetics, Iowa State University, Ames, 50011, USA.
Benbow
R M
RM
D000818:Animals; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D002467:Cell Nucleus / Q000187:drug effects* / Q000378:metabolism* / Q000648:ultrastructure; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects*; D005260:Female; D066298:In Vitro Techniques; D007399:Interphase; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013094:Spermatozoa / Q000378:metabolism; D029867:Xenopus Proteins; D014982:Xenopus laevis
D002843:Chromatin; D009687:Nuclear Proteins; D029867:Xenopus Proteins; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495891:Cdk2 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases