Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.
REFERENCES
Adachi
Y.
, Laemmli
U. K.
(1994
). Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.
EMBO J
13
, 4153
–4164
Blow
J. J.
, Laskey
R. A.
(1986
). Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.
Cell
47
, 577
–587
Blow
J. J.
, Watson
J. V.
(1987
). Nuclei act as independent and integrated units of replication in a Xenopus cell-free system.
EMBO J
6
, 1997
–2002
Blow
J. J.
, Laskey
R. A.
(1988
). A role for the nuclear envelope in controlling DNA replication within the cell cycle.
Nature
332
, 546
–548
Blow
J. J.
, Nurse
P.
(1990
). A cdc2-like protein is involved in the initiation of DNA replication in Xenopus egg extracts.
Cell
62
, 855
–862
Blow
J. J.
, Sleeman
A. M.
(1990
). Replication of purified DNA in Xenopus egg extracts is dependent on nuclear assembly.
J. Cell Sci
95
, 383
–391
Blow
J. J.
(1993
). Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor.
J. Cell Biol
122
, 993
–1002
Chen
J.
, Jackson
P. K.
, Kirschner
M. W.
, Dutta
A.
(1995
). Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA.
Nature
374
, 386
–388
Chevalier
S.
, Tassan
J.-P.
, Cox
R.
, Philippe
M.
, Ford
C.
(1995
). Both cdc2 and cdk2 promote S phase initiation in Xenopus egg extracts.
J. Cell Sci
108
, 1831
–1841
Chong
J. P. J.
, Mahbubani
M. H.
, Khoo
C.-Y.
, Blow
J. J.
(1995
). Purification of an Mcm-containing complex as a component of the DNA replication licensing system.
Nature
375
, 418
–421
Chong
J. P. J.
, Thömmes
P.
, Blow
J. J.
(1996
). The role of MCM/P1 proteins in the licensing of DNA replication.
Trends Biochem. Sci
21
, 102
–107
Fang
F.
, Newport
J.
(1991
). Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes.
Cell
66
, 731
–742
Fang
F.
, Newport
J.
(1993
). Distinct roles of cdk2 and cdc2 in RP-A phosphorylation during the cell cycle.
J. Cell Sci
106
, 983
–994
Frederick
D. L.
, Andrews
M. T.
(1994
). Cell cycle remodelling requires cell-cell interactions in developing Xenopus embryos.
J. Exp. Zool
270
, 410
–416
Gabrielli
B. G.
, Roy
L. M.
, Gautier
J.
, Philippe
M.
, Maller
J. L.
(1992
). A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2.
J. Biol. Chem
267
, 1969
–1975
Gautier
J.
, Norbury
C.
, Lohka
M.
, Nurse
P.
, Maller
J.
(1988
). Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+.
Cell
54
, 433
–439
Girard
F.
, Strausfeld
U.
, Fernandez
A.
, Lamb
N. J. C.
(1991
). Cyclin A is required for the onset of DNA replication in mammalian fibroblasts.
Cell
67
, 1169
–1179
Harland
R. M.
, Laskey
R. A.
(1980
). Regulated DNA replication of DNA microinjected into eggs of Xenopus laevis.
Cell
21
, 761
–771
Howe
J. A.
, Howell
M.
, Hunt
T.
, Newport
J. W.
(1995
). Identification of a developmental timer regulating the stability of embryonic cyclin A and a new somatic A-type cyclin at gastrulation.
Genes Dev
9
, 1164
–1176
Jackson
P. K.
, Chevalier
S.
, Philippe
M.
, Kirschner
M. W.
(1995
). Early events in DNA replication require cyclin E and are blocked by p21CIP1.
J. Cell Biol
130
, 755
–769
Knoblich
J. A.
, Lehner
C. F.
(1993
). Synergistic action of Drosophila cyclins A and B during the G2-M transition.
EMBO J
12
, 65
–74
Knoblich
J. A.
, Sauer
K.
, Jones
L.
, Richardson
H.
, Saint
R.
, Lehner
C. F.
(1994
). Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation.
Cell
77
, 107
–120
Kobayashi
H.
, Stewart
E.
, Poon
R.
, Adamczewski
J. P.
, Gannon
J.
, Hunt
T.
(1992
). Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits.
Mol. Biol. Cell
3
, 1279
–1294
Kobayashi
H.
, Stewart
E.
, Poon
R. Y. C.
, Hunt
T.
(1994
). Cyclin A and cyclin B dissociate from p34cdc2with half-times of 4 and 15 h, respectively, regardless of the phase of the cell cycle.
J. Biol. Chem
269
, 29153
–29160
Kubota
Y.
, Takisawa
H.
(1993
). Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell free extracts.
J. Cell Biol
123
, 1321
–1331
Labbe
J. C.
, Capony
J. P.
, Caput
D.
, Cavadore
J. C.
, Derancourt
J.
, Kaghad
M.
, Lelias
J. M.
, Picard
A.
, Doree
M.
(1989
). MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B.
EMBO J
8
, 3053
–3058
Labbe
J. C.
, Picard
A.
, Peaucellier
G.
, Cavadore
J. C.
, Nurse
P.
, Doree
M.
(1989
). Purification of MPF from starfish: identification as the H1 histone kinase p34cdc2 and a possible mechanism for its periodic activation.
Cell
57
, 253
–263
Lehner
C. F.
, O'Farrell
P. H.
(1990
). The roles of Drosophila cyclins A and B in mitotic control.
Cell
61
, 535
–547
Lohka
M. J.
, Masui
Y.
(1983
). Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic contents.
Science
220
, 719
–721
Luca
F. C.
, Shibuya
E. K.
, Dohrmann
C. E.
, Ruderman
J. V.
(1991
). Both cyclin A delta 60 and B delta 97 are stable and arrest cells in M-phase, but only cyclin B delta 97 turns on cyclin destruction.
EMBO J
10
, 4311
–4320
Minshull
J.
, Golsteyn
R.
, Hill
C. S.
, Hunt
T.
(1990
). The A-and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle.
EMBO J
9
, 2865
–2875
Newport
J.
(1987
). Nuclear reconstitution in vitro: stages of assembly around protein-free DNA.
Cell
48
, 205
–217
Nigg
E. A.
(1995
). Cyclin-dependent protein kinases: key regulators of the eukaryotic cell cycle.
BioEssays
17
, 471
–80
Ohtsubo
M.
, Roberts
J. M.
(1993
). Cyclin-dependent regulation of G1 in mammalian fibroblasts.
Science
259
, 1908
–1912
Pagano
M.
, Pepperkok
R.
, Verde
F.
, Ansorge
W.
, Draetta
G.
(1992
). Cyclin A is required at two points in the human cell cycle.
EMBO J
11
, 961
–971
Pagano
M.
, Pepperkok
R.
, Lukas
J.
, Baldin
V.
, Ansorge
W.
, Bartek
J.
, Draetta
G.
(1993
). Regulation of the cell cycle by the cdk2 protein kinase in cultured human fibroblasts.
J. Cell Biol
121
, 101
–111
Pines
J.
, Hunter
T.
(1990
). Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B.
Nature
346
, 760
–763
Pines
J.
, Hunter
T.
(1991
). Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport.
J. Cell Biol
115
, 1
–17
Poon
R. Y. C.
, Yamashita
K.
, Adamczewski
J. P.
, Hunt
T.
, Shuttleworth
J.
(1993
). The cdc2-related protein p40MO15 is the catalytic subunit of a protein kinase that can activate p33cdk2 and p34cdc2.
EMBO J
12
, 3123
–3132
Rempel
R. E.
, Sleight
S. B.
, Maller
J. L.
(1995
). Maternal Xenopus Cdk2-cyclin E complexes function during meiotic and early embryonic cell cycles that lack a G1 phase.
J. Biol. Chem
270
, 6843
–6855
Roy
L. M.
, Swenson
K. I.
, Walker
D. H.
, Gabrielli
B. G.
, Li
R. S.
, Piwnica Worms
H.
, Maller
J. L.
(1991
). Activation of p34cdc2 kinase by cyclin A.
J. Cell Biol
113
, 507
–514
Sheehan
M. A.
, Mills
A. D.
, Sleeman
A. M.
, Laskey
R. A.
, Blow
J. J.
(1988
). Steps in the assembly of replication-competent nuclei in a cell-free system from Xenopus eggs.
J. Cell Biol
106
, 1
–12
Shivji
M. K. K.
, Grey
S. J.
, Strausfeld
U. P.
, Wood
R. D.
, Blow
J. J.
(1994
). Cip1 inhibits DNA replication but nor PCNA-dependent nucleotide excision-repair.
Curr. Biol
4
, 1062
–1068
Strausfeld
U. P.
, Howell
M.
, Rempel
R.
, Maller
J. L.
, Hunt
T.
, Blow
J. J.
(1994
). Cip1 blocks the initiation of DNA replication in Xenopus extracts by inhibition of cyclin-dependent kinases.
Curr. Biol
4
, 876
–883
van den Heuvel
S.
, Harlow
E.
(1993
). Distinct roles for cyclin-dependent kinases in cell cycle control.
Science
262
, 2050
–2054
Vesely
J.
, Havlicek
L.
, Strnad
M.
, Blow
J. J.
, Donnella-Deana
A.
, Pinna
L.
, Letham
D. S.
, Kato
J.
, Detivaud
L.
, Leclerc
S.
, Meijer
L.
(1994
). Inhibition of cyclin-dependent kinases by purine analogues.
Eur. J. Biochem
224
, 771
–786
Walker
D. H.
, Maller
J. L.
(1991
). Role for cyclin A in the dependence of mitosis on completion of DNA replication.
Nature
354
, 314
–317
Yamashita
M.
, Yoshikuni
M.
, Hirai
T.
, Fukuda
S.
, Nagahama
Y.
(1991
). A monoclonal antibody against the PSTAIR sequence of p34cdc2catalytic subunit of maturation-promoting factor and key regulator of the cell cycle.
Dev. Growth Differ
33
, 617
–624
Zindy
F.
, Lamas
E.
, Chenivesse
X.
, Sobczak
J.
, Wang
J.
, Fesquet
D.
, Henglein
B.
, Brechot
C.
(1992
). Cyclin A is required in S phase in normal epithelial cells.
Biochem. Biophys. Res. Commun
182
, 1144
–1154
This content is only available via PDF.
© 1996 by Company of Biologists
1996
