Cdc6 protein causes premature entry into S phase in a mammalian cell-free system.
We exploit an improved mammalian cell-free DNA replication system to analyse quiescence and Cdc6 function. Quiescent 3T3 nuclei cannot initiate replication in S phase cytosol from HeLa or 3T3 cells. Following release from quiescence, nuclei become competent to initiate semiconservative DNA replication in S phase cytosol, but not in G0 phase cytosol. Immunoblots show that quiescent cells lack Cdc6 and that minichromosome maintenance (MCM) proteins are not associated with chromatin. Competence of G1 phase nuclei to replicate in vitro coincides with maximum Cdc6 accumulation and MCM protein binding to chromatin in vivo. Addition of recombinant Cdc6 to permeabilized, but not intact, G1 nuclei causes up to 82% of the nuclei to initiate and accelerates G1 progression, making nuclei competent to replicate prematurely.
Stoeber
K
K
Wellcome/CRC Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK. ks247@hermes.cam.ac.uk
Mills
A D
AD
Kubota
Y
Y
Krude
T
T
Romanowski
P
P
Marheineke
K
K
Laskey
R A
RA
Williams
G H
GH
D016475:3T3 Cells; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004261:DNA Replication; D016193:G1 Phase; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009685:Nuclear Envelope / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010539:Permeability; D011485:Protein Binding; D016192:Resting Phase, Cell Cycle; D016196:S Phase / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D018844:Cyclin-Dependent Kinases