Evidence that DNA replication is not regulated by ubiquitin-dependent proteolysis in Xenopus egg extract.
The Xenopus early embryonic cell cycle consists of rapid oscillations between mitosis and DNA synthesis. We used ubiquitin (Ub)-dependent proteolysis inhibitors to determine whether Ub-mediated proteolysis regulates the initiation of DNA replication in Xenopus egg extract. Methylated Ub, a chemically modified Ub that cannot form chains, and S5a, a Ub chain-binding subunit of the 26S proteasome, were added to extract at concentrations known to inhibit cyclin B proteolysis and their effects on cell cycle progression and DNA replication were examined. DNA replication initiated concomitant with controls and proceeded in a semiconservative fashion in the presence of both methylated Ub and S5a. However, mitotic progression was halted, showing that the inhibitors were functional. We conclude that initiation of DNA replication is not regulated by Ub-dependent proteolysis in the early Xenopus cell cycle.
Mahaffey
David T
DT
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT 84132-3201, USA.
Gorbea
Carlos
C
Rechsteiner
Martin
M
D000818:Animals; D016590:Aphidicolin / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D056765:Cyclin B2; D004261:DNA Replication; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D008745:Methylation; D009865:Oocytes / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D016601:RNA-Binding Proteins; D014020:Tissue Extracts / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D014981:Xenopus
C503758:CCNB2 protein, human; D002352:Carrier Proteins; D019926:Cyclin B; D056765:Cyclin B2; D004791:Enzyme Inhibitors; C498506:PSMD4 protein, human; D016601:RNA-Binding Proteins; D014020:Tissue Extracts; D025801:Ubiquitin; D016590:Aphidicolin; D046988:Proteasome Endopeptidase Complex