183
y.
Cell Set. Suppl. 12, 183-195 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
Nuclear structure and the control of DNA replication in the
Xenopus embryo
J.
J.
B L O W 1, M . A .
S H E E H A N 2 , J.
V . W A T S O N 3 a n d
R .
A .
L A S K E Y 2
1ICRF Cell Cycle Control Laboratory, Microbiology Unit, Department ofBiochemistry, South
Parks Road, Oxford 0X1 3QU, UK
2CRCMolecular Embryology Research Group, Department of Zoology, Downing Street,
Cambridge CB2 3EJ, UK
3MRC Clinical Oncology Unit, MRC Centre, Hills Road, Cambridge CB2 2QH, UK
Summary
W e h a v e d e v e l o p e d a c e l l - f re e s y s te m f ro m f ro g e g g s th a t e f f i c i e n tl y i n i ti a te s a n d c o m p l e te s a s i n g l e
ro u n d o f s e m i - c o n s e rv a ti v e re p l i c a ti o n . 7 0 - 1 0 0 %
D N A
m o l e c u l e s a re c o m p l e te l y re p l i c a te d
o f s p e rm
in vitro. B e f o re
c h ro m a t i n a n d u p to 4 0 %
D N A
o f p l as m id
is re p l i c a te d i t is a s s e m b l e d i n to
n u c l e i s u rro u n d e d b y a d o u b l e u n i t m e m b ra n e s tu d d e d w i th n u c l e a r p o re s . F l o w c y t o m e t ry s h o w s
th a t i n i ti a ti o n e v e n ts a re c o - o rd i n a te d w i th i n i n d i v i d u a l n u c l e i , a l th o u g h d i f f e re n t n u c l e i c a n s t a rt
to re p l i c a te a t d i f f e re n t ti m e s i n th e s a m e e x t r a c t . T h i s d e m o n s tra te s th e i m p o r t a n c e o f n u c l e a r
s t ru c t u re i n th e c o n t ro l o f D N A
re p l i c a ti o n i n th i s s y s te m .
O n l y a s i n g l e ro u n d o f s e m i - c o n s e rv a ti v e re p l i c a ti o n o c c u r s i n th e c e l l - f re e s y s te m . T h i s m i rro rs
th e w a y t h a t o n l y o n e ro u n d o f D N A re p l i c a ti o n o c c u r s i n e a c h c e l l c y c l e
in vivo. W h e n
re p l i c a te d
n u c l e i a re tra n s f e rr e d to f re s h e x t r a c t th e y a re u n a b l e to u n d e rg o a n o th e r ro u n d o f re p l i c a ti o n .
H o w e v e r, i f th e n u c l e a r e n v e l o p e is p e rm e a b i l i s e d b e f o re n u c l e i a re tra n s f e rre d to f re s h e x t r a c t , th e
D N A
b e co m e s
cap a b l e
o f u n d e rg o i n g a f u rt h e r ro u n d
o f s e m i - c o n s e rv a ti v e
re p l i c a ti o n .
T h ese
re s u l ts s u g g e s t a s i m p l e m o d e l f o r th e c o n tro l o f D N A re p l i c a ti o n w i th i n th e c e l l c y c l e , w h e re b y a n
e s s e n ti a l i n i ti a ti o n f a c t o r c a n o n l y g a i n a c c e s s to D N A
w h e n th e n u c l e a r e n v e l o p e b re a k s d o w n
d u ri n g m i to s i s .
Introduction
E u k a r y o tic
DN A
d if f e r e n t f r o m
re p l ic a tio n
is c o n tro l le d
in
a re m a r k a b le w a y , w h ic h
its b io c h e m ic a l c o u n te r p a r t, tr a n s c r ip tio n . T h e
is q u ite
in itia ti o n o f n e w
r e p li c a tio n f o rk s is s tr i c tl y p e ri o d ic , o c c u r r in g o n ly in a d is c r e te p e rio d o f th e c e ll
c y c l e . F u r th e r m o r e , in itia ti o n e v e n ts in S p h a se o n ly ta k e p la c e o n D N A th a t h a s n o t
a lre a d y b e e n r e p li c a te d
in th a t c e ll c y c l e . T h i s m e a n s th a t th e e n tir e g e n o m e is
re p l ic a te d p r e c is e l y o n c e in e a c h c e l l c y c l e . O v e r- o r u n d e r- r e p l ic a tio n o f e v e n s m a ll
s e c tio n s o f D N A
c o u ld b e f a ta l to a c e l l. H o w e v e r, v e ry li ttle is k n o w n a b o u t th e
m e c h a n is m s th a t e u k a ry o te s u se to c o n s tra in D N A
r e p li c a tio n to a s in g le , p re c is e
re p l ic a tio n o f th e g e n o m e in e a c h c e l l c y c l e .
O n e o f th e m a jo r li m ita tio n s to u n d e rs ta n d in g th e c o n tr o l o f D N A re p l ic a tio n in
e u k a ry o te s h a s b e e n th e la c k o f c e l l- f r e e s y s te m s th a t are a m e n a b le to b io c h e m ic a l
a n a ly s is . In p a rti c u l a r, it h a s b e e n d if f ic u lt to r e c r e a te th e in itia ti o n o f r e p li c a tio n
f o rk s o n d o u b le - s tr a n d e d D N A
in vitro
u s in g e u k a ry o tic c e ll e x tr a c ts . T h i s d if f ic u lty
m a y in f a c t r e f l e c t th e tig h t c o n tro l s th a t e x i s t
K e y w o rd s : D N A
re p l i c a ti o n , n u c l e a r a s s e m b l y ,
in vivo
Xenopus e g g s .
o n th e in itia ti o n p r o c e s s .
184
J. y. Blow et al.
O n e a l te rn a tiv e
is to
s tu d y
th e in itia ti o n
e v e n ts o c c u r r in g
a t v ira l o rig in s o f
re p l ic a tio n . C e ll - f r e e s y s te m s h a v e b e e n d e v e lo p e d th a t s u p p o r t a d e n o v iru s ( C h a lb e r g a n d K e l l y , 1 9 7 9 ) an d S V 4 0 ( L i an d K e l l y , 1 9 8 4 ; S ti l l m a n a n d G lu z m a n , 1 9 8 5 ;
W o bbe
et al.
1985) D N A
in vitro.
re p l ic a tio n
T h e s e s y s te m s h a v e y ie ld e d m u c h
in f o rm a tio n a b o u t th e m e c h a n ic s o f re p l ic a tio n in e u k a ry o te s . H o w e v e r, s in c e v ira l
re p l ic a tio n is c h a ra c te ri s e d
b y a m p l if ic a ti o n o f v ira l D N A
w ith in th e c e ll , th e s e
s y s te m s m a y n o t te ll u s v e ry m u c h a b o u t th e w ay th e c e l lu la r D N A
re p l ic a tio n is
c o n tr o l le d .
O u r a p p ro a c h h a s b e e n to r e c r e a te th e in itia ti o n o f c e l lu la r D N A
vitro. A s s ta rtin g
X enopus laevis.
u n d e r g o in g
th e
r e p li c a tio n
in
m a te ri a l w e h a v e u se d e g g s o f th e S o u th A f r ic a n c la w e d to a d ,
T h ese
ra p id
e g g s c o n ta i n
a la rg e
s to c k p il e
o f m a te ri a l re q u ir e d
s e r ie s o f c e l l d iv is io n s th a t o c c u r
a f te r f e r ti li s a tio n .
fo r
O ur
e x tr a c ti o n p r o c e d u re is b a s e d o n th e p io n e e rin g w o rk o f L o h k a a n d M a s u i ( 1 9 8 3 ) ,
w h o s h o w e d th a t g e n tl y h o m o g e n is e d a m p h ib ia n e g g s c o u ld s u p p o r t th e a s s e m b ly o f
s p e rm
vivo
c h r o m a ti n in to s tr u c tu r e s c lo s e ly re s e m b l in g th e m a le p ro n u c l e i f o r m e d
a f te r
f e r ti li s a tio n .
i n c o rp o r a te d
Lo hka
an d
M a s u i a lso
n o te d
th a t th e
s y n th e ti c
in
n u c le i
[ 3H ] th y m i d in e , a n d s u b s e q u e n tly e n te re d a m ito tic - li k e s ta te . T h e s e
o b s e rv a tio n s s u g g e s te d
th a t it w as p o s s i b l e to
p ro d u c e
a c e l l- f r e e
s y s te m
a m p h ib ia n e g g s c a p a b l e o f s u p p o rtin g all th e m a jo r c e l l - c y c l e a c ti v ité s
fro m
in vitro.
O u r s u b s e q u e n t w o rk s h o w e d th a t s im ila r c e l l- f r e e e x tr a c ts s u p p o rte d th e e f f ic ie n t
in itia ti o n a n d c o m p le ti o n o f a s in g le ro u n d o f s e m i- c o n s e rv a tiv e r e p l ic a tio n
in vitro.
T h e in itia ti o n o f r e p lic a tio n f o rk s in th is s y s te m is d e p e n d e n t o n th e D N A te m p l a te
b e in g a s s e m b le d in to f u n c tio n a l n u c l e i
u n d e r g o in g
a f u r th e r ro u n d
in vitro.
R e p li c a te d n u c l e i a re o n ly c a p a b le o f
o f s e m i- c o n s e r v a ti v e
re p l ic a tio n
o n c e th e i r n u c l e a r
e n v e lo p e h a s b e e n p e rm e a b i lis e d , a n e v e n t th a t n o r m a ll y o c c u r s a t m ito s i s in th e c e ll
c y c l e . T h e s e re s u lts h av e a llo w e d u s to p ro p o s e a g e n e ra l m o d e l f o r th e w ay th a t
D N A r e p l ic a tio n is c o n tro ll e d w ith in th e e u k a ry o tic c e l l c y c l e .
Materials and methods
E x t r a c t s w e re p re p a re d a s d e s c ri b e d b y B l o w a n d L a s k e y ( 1 9 8 6 ) , w i th th e m o d i f i c a ti o n s d e s c ri b e d
b y B lo w an d L ask e y (1 9 8 8 ).
F l o w c y t o m e t ry a n d u s e o f b i o ti n y l a te d d U T P w e re a s d e s c ri b e d b y B l o w a n d W a ts o n ( 1 9 8 7 ) .
D e n s i ty s u b s ti tu ti o n e x p e ri m e n ts w e re a s d e s c ri b e d b y B l o w a n d L a s k e y ( 1 9 8 6 ) .
P ro c e d u r e s
f o r re - re p l i c a ti n g
n u c l e i f o rm e d
in vitro w e re
a s d e s c ri b e d
by
B lo w
an d
L ask ey
(1 9 8 8 ).
Results
N uclear assembly in the cell-free system
On
f e r ti li s a tio n ,
th e
X enopus
eg g
e m e rg e s
fro m
its
a rr e s t
at
se c o n d
m e io ti c
m e ta p h a s e , a n d e n te r s in te rp h a s e o f th e f i rs t c e l l c y c l e . T h e m a te rn a l a n d p a te rn a l
c h ro m o s o m e s a re th e n a s s e m b le d in to p ro n u c l e i p r io r to th e o n s e t o f D N A s y n th e s is
( G r a h a m , 1 9 6 6 ) . L o w - s p e e d s u p e rn a ta n ts o f c ru s h e d e g g s c a n s u p p o rt th e a s s e m b ly
o f d e m e m b r a n a te d
s p e rm
c h ro m a ti n
in to
a p p a re n tly
n o rm a l p ro n u c l e i
( F i g . 1;
L o h k a a n d M a s u i, 1 9 8 3 ) . U n d e r th e e l e c tr o n m ic ro s c o p e , th e s e n u c le i c a n b e s e e n to
Control ofDNA replication in Xenopus
185
b e su rro u n d e d
b y a d o u b le u n i t e n v e lo p e , s tu d d e d w ith n u c le a r p o r e s ( L o h k a a n d
M a s u i,
1984;
1983,
et al.
B lo w
1987;
a c c u m u la te s o lu b le n u c le a r p r o te in s
et al.
Fo rb e s
X enopus
eg g s
is
(1983)
hav e
a s s e m b le d
sh o w n
in to
Sh e e h an
in vitro
th a t
et al.
1988).
et al.
(N ew m e y e r
b a c te r io p h a g e
s tr u c tu r e s
Su c h
n u c le i
can
1 9 8 6 b).
D N A
( ‘ p s e u d o - n u c le i’ )
m i c r o i n je c t e d
r e s e m b lin g
in to
n o rm a l
n u c le i a s s e m b le d f r o m c h r o m o s o m a l D N A . S u c h s tr u c tu r e s a re a ls o a s s e m b le d f r o m
n aked
in vitro
D N A
1 9 8 7 ; B lo w
et al.
et al.
(N ew m ey er
1 9 8 7 ; B lo w
et al.
1 9 8 6 a; B l o w
an d
L ask ey ,
1986;
N e w p o r t,
1 9 8 8 ) . P s e u d o - n u c le i a re s u rro u n d e d b y a d o u b le
u n i t e n v e lo p e s tu d d e d w ith n u c le a r p o r e s a n d lin e d b y a la y e r o f la m in s ( N e w m e y e r
et al.
1 9 8 6 a; N e w p o r t , 1 9 8 7 ) . P s e u d o - n u c l e i c a n a c c u m u l a t e s o l u b l e n u c l e a r p r o t e i n s
in vitro
(N ew m ey er
et al.
1 9 8 6 a).
Initiation o f DNA replication
Sp e rm
in v i tr o
c h r o m a t i n is e f f i c i e n t l y r e p l i c a t e d i n t h e s e e x t r a c t s ( B l o w
H u tc h is o n
et al.
te m p l a te D N A
1987).
A f te r
a p r e - s y n th e s is
is r e p lic a te d o v e r 4 - 6 h ( B lo w
la g
(B lo w
an d
L ask ey ,
1986).
ab o u t
1 h,
70-100%
o f th e
a n d L a s k e y , 1 9 8 6 ) . T h e p r e - s y n th e s is
la g c o r r e s p o n d s a p p r o x i m a t e l y t o t h e t i m e r e q u i r e d
f o r m a tio n
of
an d L ask e y , 1986;
F ig . 1
f o r c o m p le te n u c le a r e n v e lo p e
sh o w s
th a t
in c o r p o r a tio n
of
th e
th y m id i n e a n a lo g u e b i o t i n - 1 1 - d U T P o n ly o c c u r s in f u lly a s s e m b le d n u c le i ( B l o w a n d
DNA
PHASE
BI OTIN
A
0 h
B
0-5 h
C
2-5 h
D
2-5 h
+
APHI D
F i g . 1 . A s s e m b l y o f p ro n u c l e i f r o m s p e rm c h r o m a ti n i n c u b a te d in e g g e x tr a c t. D e m e m b ra n a te d
Xenopus s p e rm
n u c le i w e re i n c u b a te d in th e c e l l - f r e e e x tr a c t f o r 0 ( A ) , 0 .5 ( B ) ,
o r 2 . 5 h ( C , D ) w ith th e th y m id in e a n a lo g u e b i o t i n - l l - d U T P . N u c l e i w e re th e n is o la te d
a n d s ta i n e d w i th H o e c h s t 3 3 2 5 8 to s h o w to ta l D N A , v ie w e d u n d e r p h a s e - c o n tr a s t o p ti c s ,
o r s ta i n e d
w i th T e x a s R e d - c o n ju g a te d
s tr e p ta v id in to
sh o w
b i o ti n i n c o r p o r a ti o n in to
n a s c e n t D N A . D , e x tr a c t s u p p le m e n te d w i th 3 0 f i g m l _ 1 a p h id ic o lin ( A P H I D . ) . S c a l e
b a r , 2 0 Ju m . A l l p a n e ls a t th e s a m e m a g n i f i c a ti o n . R e p ro d u c e d f r o m
1987.
B lo w a n d W a ts o n ,
186
J. y. Blowet al.
W a ts o n , 1 9 8 7 ) . B r d U T P d e n s i ty s u b s ti tu ti o n s h o w s th a t th e
r e p r e s e n ts a s in g le c o m p le te ro u n d
in vitro
o f s e m i- c o n s e r v a tiv e r e p lic a tio n
D N A
s y n th e s is
(F ig . 2;
B lo w
a n d L a s k e y , 1 9 8 6 ) . A lth o u g h r e p lic a tio n c o n tin u e s o v e r a p e rio d o f m o r e th a n 2 h
no
s i g n if i c a n t p o p u la tio n o f r e p l i c a ti v e i n te r m e d i a te s ( w i th a d e n s i ty b e tw e e n li g h t/ li g h t
a n d h e a v y / l i g h t ) is o b s e r v e d . T h i s i m p l i e s t h a t o n g o i n g D N A
s y n th e s is
in vitto
is
d e p e n d e n t o n o n g o in g in itia tio n o f n e w r e p lic a tio n f o r k s , a c o n c lu s io n c o n f ir m e c . b y
f lo w
c y to m e tr y
F ig . 2 .
(se e b e lo w ).
B r d U T P d e n s i ty s u b s ti tu ti o n e x p e r i m e n t, s h o w in g s e m i- c o n s e r v a ti v e re p l ic a ti o n
o f s p e rm
c h r o m a ti n in th e e x tr a c t. ( • ------ • )
D e m e m b r a n a te d
Xenopus s p e rm
n u c le i
w e re in c u b a te d in e g g e x tr a c t f o r 2 ( A ) , 4 ( B ) o r 6 h ( C ) w i th B r d U T P a n d [ a '- 32P ] d A T P ,
an d D N A w a s th e n f r a c ti o n a te d o n C s C l d e n s i ty g ra d ie n ts . V e r tic a l a rro w s s h o w g ra d ie n t
d e n s i ty :
1 .7 1
( l ig h t/ li g h t,
( O ------ O ) M i n u s D N A
L L );
1 .7 5
( h e a v y / li g h t,
H L );
1 .7 9
(h eav y / h eav y ,
H H i.
c o n tr o l . In s e t to p a n e l C : a u to r a d io g ra p h o f re p l ic a te d D N A
a f te r a g a ro s e g e l e le c tr o p h o r e s is . L a n e a , c o n d i ti o n s as d e n s i ty g r a d ie n t; la n e b , p lu s
1 0 / ig m l- 1
a p h i d ic o li n ; h o r iz o n ta l a rro w , 5 0 k b D N A
and L ask e y , 1986.
m a r k e r. R e p r o d u c e d f r o m
B lo w
Control ofDNA replication in Xenopus
la c k o f h e a v y / h e a v y
The
D N A
r e p li c a te d in a s i n g le i n c u b a ti o n
in
F ig . 2 s h o w s th a t n o
in vitro.
187
sp e rm
c h r o m a tin
is r e
S i n c e r e p li c a tio n in th i s s y s te m is e f f ic ie n t,
a n d in i ti a ti o n e v e n ts o c c u r o v e r a p e r io d o f m o r e th a n 2 h , a s i g n if i c a n t a m o u n t o f r e
r e p lic a te d D N A
w o u ld b e e x p e c te d a f te r 6 h if i n i ti a ti o n e v e n ts o c c u r o n a ll s tr e tc h e s
o f D N A w ith e q u a l p r o b a b i li ty . T h e la c k o f r e - r e p li c a tio n m e a n s th a t th e e x tr a c t is in
s o m e w a y a b l e to d is tin g u i s h r e p li c a te d f r o m
u n r e p lic a te d D N A
to p e r m i t i n i ti a ti o n
o n l y o n t h e f o r m e r . T h i s m i r r o r s t h e w a y t h a t e u k a r y o t i c c e l l s c o n t r o l i n i t i a t i o n to
p r e v e n t r e - r e p l i c a ti o n in a s i n g le c e ll c y c l e .
L i k e th e i n t a c t e g g ( H a r l a n d a n d L a s k e y , 1 9 8 0 ) , th e c e l l - f r e e s y s te m a ls o r e p l i c a te s
n aked
D N A
in tr o d u c e d
in to it ( B l o w
an d
et al.
L a s k e y , 1 9 8 6 ; B lo w
1 9 8 7 ) . A f te r a
p r e - s y n th e s i s la g s i m i l a r to th a t s e e n w ith s p e r m c h r o m a t i n , p l a s m id D N A m o le c u le s
in vitro
u n d e r g o s e m i- c o n s e r v a tiv e r e p lic a tio n
w ith
sp e rm
c h r o m a tin ,
r e p lic a tio n
is
( F i g . 3 ; B lo w
u s u a lly
lim ite d
to
in c u b a tio n . G e n u in e in itia tio n ( r a th e r th a n e lo n g a tio n f r o m
d e m o n s tr a te d
by
th e
a b ility
of
th e
e x tr a c t
to
m o re e f f ic ie n tly
th a t o f s p e r m
in vitro ,
c h r o m a tin ;
a s th e y d o
in vivo
s in g le
ro u n d
in
each
p r e - f o r m e d p r i m e r s ) is
re - r e p lic a te
c ir c u m s ta n c e s ( s e e b e lo w ) . R e p lic a tio n o f n a k e d D N A
r e p lic a tio n ) th a n
an d L a sk e y , 1 9 8 6 ). A s
a
D N A
und er
c e r ta in
is le s s e f f ic ie n t ( le s s th a n 4 0 %
la r g e r n a k e d
D N A
te m p la te s r e p lic a te
( M e c h a li an d K e a rs e y , 1 9 8 4 ) .
Replication is dependent on nuclear assembly
W h e n t h e e x t r a c t is d i v i d e d i n t o s o l u b l e a n d v e s i c u l a r c o m p o n e n t s , n e i t h e r f r a c t i o n is
c a p a b le
o f a s s e m b lin g
F ig . 3.
B rd U T P
d e n s i ty
( • ------ • ) P la s m id
[ a ,- 32P ] d A T P .
n u c le i f ro m
sp e rm
s u b s tit u ti o n
of
c h r o m a tin
p las m id
(L o h k a
DN A
w a s th e n
f r a c ti o n a te d
on
M a s u i,
re p l ic a te d
p U C m S 4 w a s i n c u b a te d in e g g e x tr a c t f o r 6 h
DN A
an d
in
1984)
e x tr a c t.
w i th B r d U T P
C s C l g ra d ie n ts . V e r tic a l a rro w
an d
sh o w s
d e n s ity o f H L ( r e p l ic a te d ) D N A . ( O ------ O ) M i n u s D N A c o n tr o l . In s e t : a u to r a d io g ra p h
o f re p l ic a te d
DN A
a f te r a g a ro s e g e l e le c tr o p h o r e s is .
Lan e
a, D N A
i n c u b a ti o n ; la n e b , D N A c u t w i th m e th y l a ti o n - s e n s iti v e e n z y m e
w ith
BglI I , w h ic h lin e a ri s e s th e n a ti v e p l a s m id . H o r iz o n ta l a rro w s s h o w
o f p las m id
f o rm s
I,
II
an d
III,
and
a s is o la te d
a f te r
Dpn\\ la n e c , D N A
cu t
g e l s lo t, p o s i ti o n
p o s i ti o n o f h i g h m o le c u la r w e ig h t l in e a r D N A
( H M W L ) . R e p ro d u c e d f r o m B lo w a n d L a s k e y , 1 9 8 6 .
or
188
jf. jf. Blow et al.
n aked
D N A
( N e w p o r t,
c h r o m a tin ( S h e e h a n
1 9 8 7 ); an d
et al.
n e ith e r f r a c tio n is c a p a b le o f r e p li c a tin g s p e im
1988) o r n aked D N A
( N e w p o r t, 1 9 8 7 ) . O n r e c o m b in a tio n
o f t h e s o l u b l e a n d v e s i c u l a r f r a c t i o n s , a ll t h e s e a c t i v i t i e s a r e r e c o n s t i t u t e d . F u r t h e r ,
u se
o f th e
th y m id in e
a n a lo g u e
p la c e in n u c le i a s s e m b le d f r o m
b io tin - 1 1 - d U T P
sp e rm
a n d in p s e u d o - n u c le i a s s e m b le d f r o m
s h o w s th a t D N A
s y n th e s i s ta k e s
c h r o m a tin ( F i g . 1 ; B lo w
a n d W a ts o n , 1 9 8 7 ) ,
et al.
1 9 8 8 ) . T h e s e r e s u i ts
n aked D N A
( B lo w
s u g g e s t th a t f o r D N A to b e r e p l i c a te d in e g g c y to p l a s m i t m u s t f ir s t b e a s s e m b le d in to
a n u c le u s .
To
in v e s tig a te
r e p lic a te d
W a ts o n ,
in
p o in t
f u r th e r ,
p r e s e n c e o f th e
D N A
in e g g e x tr a c t w ith
h as
r e p lic a te d ,
an d
w e
p e rfo rm e d
th y m id in e
1 9 8 7 ) . F ig . 4 , p a n e ls a a n d
1 . 5 h in c u b a tio n
sp e rm
th is
th e
f lo w
c y to m e tr y
a n a lo g u e b i o t i n - l l - d U T P
b , s h o w s a p o p u la tio n o f s p e r m
on
n u c ie i
( B lo w
an d
n u c le i a f te i
a
b i o t i n - l l - d U T P . A t th i s s ta g e a b o u t h a lf t a e
ab o u t
h a lf
th e
n u c le i
hav e
in c o r p o r a te d
b io tin
( F i g . 4 B ) . F i g . 4 C s h o w s th i s p o p u la tio n o f n u c l e i a n a ly s e d b y f lo w c y to m e tr y . T l e
sp e rm
n u c le i, in itia lly
h a p lo id , r e p lic a te
D N A
u n til th e y
h a v e a c h ie v e d
a d ip lo id
D N A c o n t e n t . T h e y in c o r p o r a te b i o t i n in p r o p o r ti o n to t h e e x t e n t o f r e p l i c a ti o n th s y
h a v e u n d e r g o n e . H o w e v e r , o n c e th e y h a v e r e a c h e d th e d ip lo id D N A
Number
BI O T I N
F ig . 4 .
c o n te n t, D N A
F l o w c y to m e tr i c a n a l y s is o f s p e r m c h r o m a ti n re p l ic a te d
b io tin y l a te d
d U TP.
e x tr a c t f o r l . S h
D e m e m b r a n a te d
Xenopus
s p e rm
n u c le i
invitro in th e p r e s e n c e o f
w e re
in c u b a te d
in
eg g
w i th b i o t i n - 1 1 - d U T P . N u c l e i w e re is o la te d a n d s ta in e d w i th a D N A -
b in d i n g f l u o ro c h r o m e ( A ) an d f l u o r e s c e n t- la b e l le d s tr e p ta v id in ( B ) , a n d th e n a n a ly s e d b y
f lo w c y to m e tr y ( C ) . A , B . F l u o r e s c e n c e m ic r o s c o p y o f s ta in e d n u c le i. S c a l e b a r , 5 0 ^ m .
A p p ro x im a te ly 5 0 %
o f th e te m p l a te D N A
h a s b e e n r e p l ic a te d d u ri n g th e i n c u b a ti o n .
C . T h e p o p u l a tio n o f n u c l e i is d is p la y e d in th e f o r m o f a c o n to u r p l o t; a x e s re p re s e n t
b io tin a n d D N A c o n te n ts o f in d iv id u a l n u c le i. I N a n d 2 N g iv e h a p lo id a n d d ip l o id D N A
c o n te n ts . S ta n d a rd
s in g l e - d im e n s i o n a l f r e q u e n c y p lo ts f la n k th e c o n to u r p l o t.
d u c e d f r o m B lo w a n d W a ts o n , 1 9 8 7 .
R e p ro
189
Control ofDNA replication in Xenopus
s y n th e s is a n d b io tin in c o r p o r a tio n c e a s e , c o n s is te n t w ith th e d e n s ity s u b s titu tio n
e x p e ri m e n ts ( F i g . 2 ) .
M o s t n u c le i in
F ig . 4 C
a re e ith e r h a p lo id
o r d ip lo id , w ith
a re la ti v e ly
s m a ll
p ro p o rtio n in ‘ S p h a s e ’ in b e tw e e n . T h i s s h o w s th a t in d iv id u a l n u c l e i re p l ic a te
re la tiv e ly f a s t
in vitro
(1 h o n a v e r a g e ) . H o w e v e r, d i f f e re n t n u c l e i s ta r t to re p l ic a te
( ‘ e n te r S p h a s e ’ ) a t d if f e r e n t tim e s o v e r m o r e th a n 1 .5 h , a l th o u g h th e y a ll s h a re th e
s a m e c y to p la s m i c e n v ir o n m e n t. T h i s is c o n s is te n t w ith d e n s ity s u b s titu tio n e x p e r
im e n ts ( e .g . F i g . 2 ) th a t s h o w in itia ti o n o c c u r r in g o v e r a p e rio d g r e a te r th a n 2 h . T h e
a s y n c h ro n y
o f e n tr y
in to
S p h a se is lik e ly to
be
d u e to
a s y n c h ro n y
o f n u c le a r
f o r m a tio n ( B l o w a n d L a s k e y , 1 9 8 6 ) .
P u l s e la b e l li n g n u c le i w ith b i o tin - 1 1 - d U T P s h o w s th a t as n u c l e i e n te r S p h a se ,
e a c h o n e u n d e rg o e s a b u r s t o f a b o u t
in itia tio n s ( B l o w
a n d W a ts o n ,
e x tr a c t u n d e rg o
th is
1 0 0 0 0 0 s y n c h ro n o u s o r n e a r- s y n c h ro n o u s
1 9 8 7 ) . H o w e v e r, d i f f e re n t n u c l e i in th e s a m e e g g
b u r s t o f in itia ti o n s
a t d if f e r e n t tim e s .
So m e
s ig n a l m u s t
a p p a re n tly a c t lo c a l ly th ro u g h o u t a s in g le n u c l e u s to c o - o rd in a te th is b u r s t. T h i s
s ig n a l c o u ld p la u s ib ly b e th e a c c u m u la tio n o f a s o l u b le n u c l e a r p r o te in w i th in th e
n u c le u s o n c e n u c l e a r e n v e lo p e f o rm a tio n is c o m p le te . T h e s e r e s u lts d e m o n s tr a te th a t
n u c le a r o r g a n is a tio n is a f u n d a m e n ta l c o m p o n e n t o f D N A r e p lic a tio n c o n tr o l in th is
s y s te m .
Entry into mitosis
in v itro
W h e n th e c e l l - f r e e e x tr a c t is p e r m itte d to s y n th e s is e p ro te in
in vitro,
it is c a p a b l e o f
p a s s in g in to a m ito s i s - li k e s ta te ( L o h k a a n d M a s u i, 1 9 8 3 ; H u tc h is o n
et al.
1987,
1 9 8 8 ; B lo w a n d L a s k e y , 1 9 8 8 ) . F i g . 5 s h o w s a ty p ic a l tim e c o u r s e o f th e s e e v e n ts
vitro.
in
M o s t n u c l e i h a v e b e e n a s s e m b le d b y 1 h ( F i g . 5 a - d ) , a n d D N A s y n th e s is th e n
o c c u rs o v e r th e n e x t 2 h ( F i g . 5 e - h ) . I f p r o te in s y n th e s is is p re v e n te d b y c y c l o h e x is ta te ( F i g . 5 i , j) . H o w e v e r, if
p r o te in s y n th e s is is p e rm itte d
Gz ( i n te r p h a s e )
in vitro, a f te r 4 - 5 h th e
n u c l e a r e n v e lo p e
( F i g . 5 k , 1).
an d
m id e , r e p li c a te d
n u c le i re m a in in a
d is a p p e a rs
c h r o m a ti n c o n d e n s e s a n d th e
H u tc h is o n
c o lle a g u e s h a v e
e x tr a c t p re p a ra tio n so th a t e x tr a c ts c a n p a s s th ro u g h s e v e ra l c e ll c y c l e s
( H u tc h i s o n
et al.
re f in e d
in vitro
1 9 8 7 , 1 9 8 8 ) . T h e s e e x tr a c ts c a n o s c illa te b e tw e e n in te rp h a s e an d
m ito s is o n a ti m e s c a le v e ry c lo s e to th a t o f th e o s c illa tio n
( 1 9 8 9 ) h a v e s h o w n th a t s y n th e s is o f c y c l in p r o te in
in vitro
in vivo.
M in s h u ll
et al.
is r e q u ir e d f o r th e e x tra c t
to p a s s f ro m in te rp h a s e in to m ito s is .
The prevention o f re-replication
W h y d o e s n ’t D N A re p l ic a te a s e c o n d tim e in th e c e l l- f r e e s y s te m ? F i g . 6 s h o w s th a t
th is is d u e to s o m e f e a tu re o f r e p li c a te d n u c l e i ( B l o w
e x p e ri m e n t s p e r m n u c l e i w e re in c u b a te d
an d
in c u b a te d
in f r e s h
e x tr a c t.
in vitro
B rd U T P
a n d L a s k e y , 1 9 8 8 ) . In th is
f o r v a rio u s p e r io d s , th e n is o la te d
d e n s i ty
s u b s titu tio n w as th e n u se d
to
d e te r m in e w h e th e r D N A th a t re p li c a te d in th e f i rs t e x tr a c t c o u ld b e r e - r e p li c a te d in
th e s e c o n d e x tr a c t. W h e n in ta c t n u c l e i a re tra n s f e rr e d f ro m e x tr a c t in in te rp h a s e , th e
re p lic a te d D N A
is in c a p a b le o f re - r e p li c a ti o n in f r e s h e x tr a c t. H o w e v e r, o n c e th e
e x tr a c t h a s p a s se d in to m ito s i s , th e c h ro m o s o m e s tra n s f e rr e d to f r e s h e x tr a c t b e c o m e
190
y. y. Blowet al.
u n d e rg o in g
c a p a b le
of
( F ig . 6,
7A ).
N u c le i
s y n th e s i s
in h ib ito r
(M in s h u ll
et al.
a n o th e r
fro m
c o m p le te
e x tr a c ts
c y c lo h e x im id e
1 9 8 9 ) , re m a in
ro u n d
b lo c k e d
( F ig . 5)
of
in
or
s e m i- c o n s e r v a tiv e
in te r p h a s e
by
e ith e r
d e s tr u c tio n
in c a p a b le o f r e - r e p lic a tio n
of
in f r e s h
re p lic a tio n
by
th e
p r o te in
c y c lin
m RN A
e x tr a c t ( F ig . 6 ,
7B ).
A
n u m b e r o f d i f f e r e n t c h a n g e s o c c u r to
D N A
d u r in g
m ito s is , s u c h
as n u c le a r
e n v e lo p e b re a k d o w n , c h r o m a tin c o n d e n s a tio n a n d h is to n e p h o s p h o r y la tio n . F ig . 7
s h o w s e x p e r i m e n t s t o d e t e r m i n e w h i c h o f t h e s e m i t o t i c f e a t u r e s is c a p a b l e o f a l l o w i n g
D N A
in vitro
to r e - r e p l i c a t e i n t h e c e l l - f r e e s y s t e m . S p e r m c h r o m a t i n w a s r e p l i c a t e d
in th e p r e s e n c e o f c y c l o h e x im i d e to m a in ta in th e D N A
in in te r p h a s e n u c le i. T h e s e
n u c le i w e r e th e n is o l a te d , g iv e n v a r i o u s t r e a tm e n ts , a n d th e n a s s a y e d f o r th e i r a b i lity
to
r e - r e p lic a te
in
fre sh
n u c le a r e n v e lo p e , s u c h
e x tr a c t.
T r e a tm e n ts
as p u n c tu r in g
th a t
cau sed
it w ith l y s o l e c i th i n
p e r m e a b ilis a tio n
of
th e
( F i g . 7 D ) o r d ig e s tin g it
w ith p h o s p h o li p a s e ( F i g . 7 E ) , p e r m i tte d a s i g n i f i c a n t n u m b e r o f i n te r p h a s e n u c le i to
re - r e p lic a te
in
fre sh
e x tr a c t.
T r e a tm e n ts
th a t
do
not
p e rm e a b ilis e
th e
n u c le a r
e n v e lo p e , s u c h a s p h o s p h a ta s e tr e a tm e n t ( F i g . 7 F ) , le a v e th e n u c le i s till u n a b le to r e
r e p li c a te . T h e p e r m e a b i li s e d n u c le i a re r e - a s s e m b le d in th e f r e s h e x tr a c t b e f o r e r e
r e p li c a tio n o c c u r s . T h e s e r e s u l ts s h o w th a t th e p e r io d ic a s s e m b ly a n d d is a s s e m b ly o f
t h e n u c l e a r e n v e l o p e p l a y s a m a jo r p a r t i n c o n t r o l l i n g D N A
r e p li c a tio n w ith in th e
e u k a r y o ti c c e ll c y c l e .
Con trol
( —c y c l o h e x i m i d e )
DNA
+ C y c lo h e x im id e
DNA
a
*•
1h
#
b
1“ ■ o
k
■ L>
e
c
C
1 1 1
’
I
g
r
f
u
h
•
■
*
F ig . 5 .
n a te d
5h
/
J
•
V
.
j
5
’
rV /- . O
w*
f
\
h
.
Phase
V .
.
4
.
3
s
Phase
k
'
/•
4
r
_
P r o te i n s y n th e s is p e r m i ts e x tr a c t to p r o g re s s in to a m i to s i s - l i k e s ta te . D e m e m b r a -
Xenopus s p e rm
n u c le i w e re in c u b a te d in e g g e x tr a c t f o r 1 ( a , b , c , d ) , 3 ( e , f , g , h ) , o r
( i , j, k , l ) , p l u s ( c , d , g , h , k , l ) o r m i n u s ( a , b , e , f , i , j)
1 0 0 ,u g m l _ 1 c y c l o h e x i m i d e . N u c le i
w e re v ie w e d w e t b y H o e c h s t 3 3 2 5 8 U V f l u o r e s c e n c e ( a , c , e , g , i , k ) o r b y p h a s e c o n tr a s t
o p ti c s ( b , d , f , h , j, l ) . S c a l e b a r , 2 0 ,u m .
<
d
191
Control ofDNA replication in Xenopus
Discussion
E x tr a c ts o f
and
M asu i
X enopus
e g g s , p r e p a r e d b y m e t h o d s s i m i l a r to t h o s e d e s c r i b e d b y L o h k a
( 1 9 8 3 ) , s u p p o r t th e
D N A
in vitro
1980;
M e c h a li
( B lo w
an d
an d
in itia tio n
of D N A
1987).
B e fo re
1984),
th e
eg g
in itia tio n
o c c u rs,
1 9 8 7 ; B lo w
R e p lic a te d
d o u b l e - s tr a n d e d
can
r e p lic a te
th e
te m p la te
( B lo w
an d L ask e y ,
p la s m id
D N A
a n d L a s k e y , 1 9 8 6 ; B lo w
m u st
be
a s s e m b le d
in to
a
a n d L a s k e y , 1 9 8 6 ; N e w p o r t, 1 9 8 7 ;
1987; Sh eeh an
et al.
1 9 8 8 ; B lo w
et al.
1988).
n u c le i a re n o t c a p a b le o f r e - r e p li c a tio n u n til th e n u c le a r e n v e lo p e h a s
b e e n p e rm e a b ilis e d
re p lic a tio n
a n d W a ts o n ,
e x tr a c t
s e q u e n c e s ( B lo w
f u n c tio n a l n u c le u s b y th e c e l l - f r e e s y s te m
H u tc h is o n ,
on
L a s k e y , 1 9 8 6 ) . L ik e th e in ta c t e g g ( H a r la n d
K e a rse y ,
m o le c u le s w ith o u t r e q u ir in g s p e c ia l D N A
et al.
r e p lic a tio n
in s o m e w a y ( B lo w
is c o n tr o l l e d
a n d L a s k e y , 1 9 8 8 ) . T h i s im p lie s th a t D N A
in th i s s y s te m
by
f a c to r s b e tw e e n n u c le u s a n d c y to p l a s m . A
th e c o m p a r tm e n ta li s a ti o n o f r e p li c a tio n
s im p le m o d e l f o r h o w
t h i s c o u l d w o r k is
o u tlin e d in F i g . 8 .
A n
e s s e n ti a l in i ti a ti o n
w h e n it is a d d e d
f a c to r ( s )
w h ic h
to t h e e g g e x t r a c t ;
w e c a ll L ic e n c in g
in vivo,
th is w o u ld
F a c t o r b i n d s to
D N A
h a p p e n w h e n th e n u c le a r
e n v e lo p e is b r o k e n d o w n a t m i to s i s ( F i g . 8 A ) . In i ti a ti o n o f r e p l i c a ti o n d o e s n o t o c c u r
a t th i s s ta g e s i n c e th e te m p l a te D N A
i s n o t i n a n u c l e u s . T h e n e x t s t a g e ( F i g . 8 B ) is
n u c le a r a s s e m b l y . L i c e n c i n g F a c t o r is u n a b l e to c r o s s th e n u c l e a r e n v e lo p e , s o th a t it
is
p re se n t
in
th e
n u c le u s
o n ly
w h e re
bound
to
D N A .
A t so m e
s ta g e
a f te r
T im e o f tr a n s f e r ( h )
F i g . 6 . T h e a b il ity o f re p l ic a te d D N A to r e - r e p l ic a te w h e n a d d e d to f r e s h e x tr a c t c h a n g e s
w ith ti m e a n d
d e p e n d s o n p r o te in s y n th e s is . D e m e m b r a n a te d
Xenopus s p e rm
n u c le i
w e re in c u b a te d in e g g e x tr a c t w ith B r d U T P a n d [ar-32P ] d A T P f o r 1 - 5 h ; n u c l e i w e re th e n
is o la te d a n d a d d e d to f r e s h e x tr a c t c o n ta in i n g B r d U T P , w h e re th e y w e re in c u b a te d f o r a
f u r th e r
5 h.
DN A
w as
is o la te d
and
f r a c ti o n a te d
on
C sC l
d e n s i ty
g ra d ie n ts .
The
p r o p o rtio n o f re - r e p l ic a te d ( H H ) D N A w a s th e n m e a s u r e d . ( ♦ - - - - - - - ♦ ) F i r s t a n d s e c o n d
e x tr a c ts
c o n ta in e d
1 0 0 / Jg m l -
c y c lo h e x im id e .
(El------ □
)
Firs t
an d
u n tr e a te d a n d a c tiv e in p ro te in s y n th e s is . E i th e r f i rs t ( • ) , o r s e c o n d
c o n ta in e d c y c l o h e x i m i d e . R e p ro d u c e d f r o m B lo w a n d L a s k e y , 1 9 8 8 .
se c o n d
e x tr a c ts
( O ) e x tr a c ts o n ly
th e
192
jf. J. Blow et al.
o f n u c le a r a s s e m b ly , s o m e s ig n a l s p r e a d s th r o u g h o u t th e
c o m p le tio n
e n v iro n m e n t
(F ig . 8 C )
c a u s in g
L ic e n c in g F a c to r ( F i g . 8 D ; B lo w
s u p p o r tin g
a
s in g le
in itia tio n
T h e r e f o r e o n c e th e D N A
a
b u rst
of
in itia tio n
e v e n ts
at
in tr a n u c b a r
s i te s
of
bound
a n d W a ts o n , 1 9 8 7 ) . L i c e n c i n g F a c to r is c a p a b le o f
e v e n t,
a f te r
w h ic h
it
is
in a c tiv a te d
or
d e s tr o y e d .
w ith i n th e n u c l e u s h a s f u l ly r e p l i c a t e d , th e r e is n o m o r e
L i c e n c i n g F a c to r w ith in th e n u c le u s . L i c e n c i n g F a c t o r s till p r e s e n t in th e c y to p la s m
c a n n o t g a in a c c e s s to t h e D N A
u n til th e n u c le a r e n v e lo p e is p e r m e a b ilis e d , a p r o c e s s
n o r m a ll y ta k in g p la c e d u r in g m i to s is .
S lig h tly
m o r e c o m p lic a te d
v e r s io n s o f th is c o m p a r tm e n ta lis a tio n
th e o r y
can
be
d e v is e d w h e r e b y a n in h ib ito r o f r e p lic a tio n b u ild s u p in th e n u c le u s , a n d c a n o n ly b e
.
P ro te in
sy n th e sis
A
X
1
V
i
.
l
*
*
i
<
z
Q
•o
.
HH
HL
1
1
C
C y c lo h e x im id e +
—1_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ f r - r w - - 0
- - - - - - - - - - - - -- - - - - - - - - - - - -
i
A
l
HL
.
.
HH
B
C y c lo h e x im id e +
M o c k tre a tm e n t
'
iu
.
HH
HL
.
1
1
D
.
C y c lo h e x im id e +
L y so le c ith in
M PF
r^T-- ---
i
V r i1a-x- D
- -U
- -JU
- - -r)U>i'WW — ,——
' -----
HH
HL
^
^
HL
HH
E
;
F
i
C y c lo h e x im id e +
g
V
M e littin /
P h o sp h o lip ase
C y c lo h e x im id e +
P h o sp h atase
‘V
^
r r i v
n m
n x x y i ^
.
Fra c tio n n o .
F ig . 7 .
N u c l e a r e n v e lo p e p e rm e a b i l is a ti o n b e tw e e n i n c u b a tio n in tw o s u c c e s s iv e e x tr a c ts
a llo w s r e - r e p l i c a ti o n o f re p l ic a te d n u c le i ( s e e n a s H H
D N A ) . ( A ) N u c l e i re p l ic a te d in
e x tr a c t a c ti v e in p r o te in s y n th e s is . ( B - F ) N u c l e i r e p lic a te d in th e p r e s e n c e o f c y c l o h e x i m id e , a n d tr e a te d w i th : B , m o c k - tr e a tm e n t; C , m a tu r a ti o n - p r o m o ti n g f a c to r ; D , ly so l e c i th i n ;
E,
m e l l i ti n
an d
p h o s p h o l ip a s e ;
F,
p h o s p h a ta s e .
The
p e r c e n ta g e
of
to ta l
32P - l a b e ll e d D N A in e a c h f r a c ti o n is g iv e n . R e p ro d u c e d f r o m B lo w a n d L a s k e y , 1 9 8 8 .
Control ofDNA replication in Xenopus
193
re le a s e d b y n u c le a r e n v e lo p e p e r m e a b ilis a tio n . H o w e v e r , th e m o d e l p r e s e n te d
( F ig . 8)
is th e
s im p le s t o n e th a t c a n
a c c o u n t f o r a ll th e
c a p a b le o f a c c o u n tin g f o r a n u m b e r o f o b s e r v a tio n s m a d e
W h e n G i an d G
o f in itia tin g
2
D N A
d a ta . T h i s m o d e l a ls o
c e lls a re f u s e d , th e G i n u c le u s , b u t n o t th e G
2
(R ao
1 9 8 0 ) . T h i s r e s u lt w o u ld
b e p r e d ic te d
h y b rid
fro m
e n te r s
is
in vivo.
s y n th e s is b e f o r e
th e
h e re
m ito s is
n u c le u s is c a p a b le
an d
Jo h n s o n ,
o u r th e o r y , s in c e o n ly th e G j n u c le u s
( F i g . 8 B ) a n d n o t th e G 2 n u c le u s ( F i g . 8 E ) c o n ta in s L ic e n c in g F a c to r . S im ila r ly , G
2
H e L a c e ll n u c l e i m u s t b e p r e p a r e d u s i n g d e te r g e n t in o r d e r f o r th e m to b e c a p a b l e o f
r e p l i c a t i o n i n t h e f i r s t c e l l c y c l e a f t e r m i c r o i n je c t i o n i n t o
al.
X enopus
eg g s (D e R o ep er
et
1977).
N o t a ll e u k a r y o te s u n d e r g o f u ll n u c le a r e n v e lo p e b r e a k d o w n a t m i to s is . H o w e v e r ,
th e y c o u ld s till c o n t r o l D N A r e p l i c a ti o n in th e m a n n e r s u g g e s te d a b o v e if th e i r n u c le i
u n d e rg o
a tr a n s ie n t p e r m e a b ilis a tio n
+
+
+
+
to
+
a L ic e n c in g
+
+
F a c to r
d u r in g
m ito s is .
P o s-
+
+
B
o
C/ 3
03
D
Fig . 8 .
M o d el
(A ) L ic e n c in g
fo r
th e
F a c to r
c o n tr o l
(+ ),
of
b in d s
DN A
to
r e p l ic a ti o n
DN A .
(B)
in
DN A
th e
Xenopus
e a rly
e m b ry o .
is
a sse m b le d
i n to
n u c le u s .
( C ) In i ti a ti o n a t l ic e n s e d s ite s o c c u r s c o o rd in a te l y th r o u g h o u t i n d i v i d u a l n u c le i ( B l o w
a n d W a ts o n , 1 9 8 7 ) . ( D ) L i c e n c i n g F a c to r is i n a c ti v a te d b y i n iti a ti o n o r th e p a s s a g e o f a
re p l ic a ti o n
fo rk .
(E)
Fu lly
r e p lic a te d
DN A
c a n n o t r e - r e p l ic a te
due
to
e x c lu s io n
of
L i c e n c i n g F a c t o r f r o m D N A b y th e n u c le a r e n v e lo p e . B r e a k d o w n o f th e n u c le a r e n v e lo p e
d u ri n g m i to s i s a llo w s a c c e s s o f th e L i c e n c i n g F a c to r to th e D N A , to p r e p a re i t f o r D N A
s y n th e s is in th e n e x t c e ll c y c l e . R e p ro d u c e d f r o m B lo w a n d L a s k e y , 1 9 8 8 .
194
J. jf. Blow et al.
o f a n u c le a r
s e s s io n
e n v e lo p e ,
th e
u se
o f m u l tip le
r e p li c a tio n
o r ig in s a n d
th e
p re v e n tio n o f re - r e p li c a ti o n in a s in g le c e l l c y c l e all s e e m to b e g e n e ra l f e a tu r e s o f
e u k a r y o te s .
C o m p a rtm e n ta li s a tio n o f e s s e n tia l r e p lic a tio n f a c to rs d u rin g th e c e ll
c y c le p ro v id e s a p o s s i b l e e x p l a n a tio n o f w h y th e s e f e a tu re s all o c c u r to g e th e r.
J. J. B . i s a B e i t M e m o ri a l F e l l o w . T h i s w o rk w a s s u p p o rte d b y th e C a n c e r R e s e a rc h C a m p a i g n .
References
Bl
o w
,
J. , D il w o r t h ,
J.
M ., D in g
S.
,
wall
M il
C .,
l s, A .
D.
La s k ey,
and
R.
A .
(1 9 8 7 ).
Xenopus e g g s . Phil. Trans. R. Soc. Lond. B
C h ro m o s o m e re p l i c a ti o n i n c e l l - f re e s y s te m s f r o m
317, 4 83-494.
Bl
o w
,
La s k ey,
J. J. a n d
R . A . ( 1 9 8 6 ) . I n i ti a ti o n o f D N A re p l i c a ti o n i n n u c l e i a n d p u ri f i e d D N A
Xenopus e g g s . Cell 4 7 ,
b y a c e l l - f re e e x t r a c t o f
Bl
o w
,
J.
La s k ey,
and
J.
R. A .
(1 9 8 8 ).
o w
,
J.
Sh e e h a n ,
J. ,
re p l i c a ti o n
in
M .
c e l l - f re e
W a t so n ,
A .,
577-587.
ro l e f o r th e n u c l e a r e n v e l o p e
Nature, Lond. 3 3 2 ,
re p l i c a ti o n w i th i n th e c e l l c y c l e .
Bl
A
e x tra c ts
J.
La s k ey ,
and
V .
Xenopus
of
i n c o n tro l l i n g
D N A
546-548.
R.
A .
Cancer Cells
eg g s.
(1 9 8 8 ).
6
R e g u l a te d
(C o l d
S p ri n g
D N A
H a rb o r
L a b o ra to ry ) , 2 9 7 - 3 0 3 .
Bl
o w
,
J.
Ch a l
,
l ber g
W a t s o n , J.
and
J.
Acad. Sci. U.S.A. 7 6 ,
D e Ro
e pe r
D N A
,
Kel
D . and
M .
(1 9 8 7 ).
D N A
l y, T .
N u cl e i
act
as
in d e p e n d e n t
re p l i c a ti o n s y s te m .
(1 9 7 9 ).
an d
EMBOJ. 6 ,
A d e n o v i ru s D N A
i n te g ra te d
u n i ts
of
1997-2002.
re p l i c a ti o n
in vitro. Proc. natn.
655-659.
S m it h ,
A .,
V .
Xenopus c e l l - f re e
re p l i c a ti o n i n a
W a t t , R.
J. A . ,
s y n th e s i s i n G i a n d G
2
Ba r r y ,
A . and
J. M . ( 1 9 7 7 ) . C h ro m a t i n d i s p e rs a l a n d
H e L a c e l l n u c l e i i n j e c te d i n to
Xenopus e g g s . Nature, Lond. 2 6 5 ,
469-470.
Fo r b e s , D .
K ir s c h n e r ,
J. ,
N e w po r t ,
M . W . and
l i k e s t ru c t u re s a ro u n d b a c te ri o p h a g e D N A
G r a h a m,
C . F . ( 1 9 6 6 ) . T h e re g u l a ti o n o f D N A
J. Cell Sci. 1 ,
Har l and,
egg s
L a s k ey,
oiX. laevis. Cell 2 1 ,
D N A
R . A . ( 1 9 8 0 ) . R e g u l a te d re p l i c a ti o n o f D N A
D r e pa u l ,
C . J. , C o x , R . ,
Fo r d ,
C . J. , C o x , R . a n d
R . S .,
Kel
l y,
T.
G o m pe r t s ,
M . and
Xenopus e g g s . EMBOJ. 6 ,
m i c ro i n j e c te d i n to
Fo r d ,
C . C . ( 1 9 8 7 ) . P e ri o d i c
2003-2010.
C . C . ( 1 9 8 8 ) . T h e c o n t ro l o f D N A re p l i c a ti o n i n a c e l l - f re e
e x t r a c t t h a t re c a p i tu l a te s a b a s i c c e l l c y c l e
L i , J. J. a n d
13-23.
761-771.
s y n th e s i s i n c e l l - f re e e x t r a c t s o f
H u t c h is o n ,
Xenopus e g g s . Cell 3 4 ,
s y n th e s i s a n d m i to s i s i n m u l ti n u c l e a te f ro g e g g s .
363-374.
R. M . and
H u t c h is o n ,
J. W . ( 1 9 8 3 ) . S p o n ta e o u s f o rm a ti o n o f n u c l e u s
m i c ro i n j e c t e d i n to
in vitro. Development 1 0 3 , 5 5 3 - 5 6 6 .
in vitro. Proc. natn. Acad. Sci. U.S.A.
( 1 9 8 4 ) . S i m i a n v i ru s 4 0 re p l i c a ti o n
81, 6973-6977.
Lo hka , M.
M a s u i,
and
J.
Y .
(1 9 8 3 ).
F o rm a ti o n
in vitro o f s p e rm p ro n u c l e i
Science 2 2 0 , 7 1 9 - 7 2 1 .
an d
m i to ti c
c h ro m o s o m e s i n d u c e d b y a m p h i b i a n o o p l a s m i c c o n te n ts .
Lo h ka , M.
and
J.
M a s u i, Y .
e n v e lo p e as se m b ly an d
eg g s.
J. Cell Biol. 9 8 ,
M e c h a l i, M .
and
inXenopus e g g s
M in s h u l l ,
Bl
J. ,
(1 9 8 4 ).
R o les
of
c y to s o l
o w
D . D .,
,
J.
n u cle i
a c c u m u l a ti o n .
N e w po r t , J.
Cell 4 8 ,
R a o , P.
p a rti c l e s
in
n u cle ar
am p h ib ian
S . ( 1 9 8 4 ) . L a c k o f s p e c i f i c s e q u e n c e re q u i re m e n t f o r D N A re p l i c a ti o n
and
Hu n t ,
Xenopus e g g s
Fi n l
T .
(1 9 8 9 ).
T ra n s l a t i o n
to e n te r m i to s i s .
a y, D . R . and
N e w m e y e r , D . D ., L u c o c q ,
of
c y to p l a s m i c
c o m p a re d w i th h i g h s e q u e n c e re q u i re m e n t i n y e a s t.
Fo r bes ,
Cell 5 6 ,
D . J.
a c ti v e
EMBOJ. 5 ,
J. M . ,
in
Bu r g l
n u cle ar
in , T .
p ro te i n
R.
o f cy clin
Cell 3 8 ,
m RN A
55-64.
i s n e c e s s a ry f o r
947-956.
In vitro t ra n s p o rt o f f l u o re s c e n t
J. Cell Biol. 1 0 3 , 2 0 9 1 - 2 1 0 2 .
(1 9 8 6 b ).
n u c l e a r p ro te i n a n d e x c l u s i o n o f n o n - n u c l e a r p ro te i n s .
in vitro
an d
p ro n u c l e a r f o rm a ti o n i n c e l l - f re e p re p a ra ti o n s f ro m
1222-1230.
K ea r s ey ,
e x t r a c t s o f a c ti v a te d
New m eyer ,
s p e rm
and
D e R o b e r t is ,
tra n s p o r t :
A TP
is
E . M . (1 9 8 6 a ). A ssem b ly
re q u i re d
for
n u cle o p las m in
501-510.
( 1 9 8 7 ) . N u c l e a r re c o n s t i tu t i o n
in vitro-, s ta g e s o f a s s e m b l y a ro u n d
p ro te i n - f re e D N A .
205-217.
N . and
Jo h n s o n , R. T .
s y n th e s i s a n d m i to s i s .
( 1 9 8 0 ) . M a m m a l i a n c e l l f u s i o n : s tu d i e s o n th e re g u l a ti o n o f D N A
Nature, Lond. 2 2 5 ,
159-164.
Control ofDNA replication in Xenopus
195
S h e e h a n , M . A . , M i l l s , A . D . , S l e e m a n , A . M ., L a s k e y , R . A . a n d B l o w , J. J. ( 1 9 8 8 ) . S te p s in
th e a s s e m b l y o f re p l i c a ti o n - c o m p e te n t n u c l e i i n a c e l l - f re e s y s te m
Biol. 1 0 6 ,
f ro m
Xenopus e g g s . J. Cell
1-12.
S t i l l m a n , B . W . a n d G l u z m a n , Y . ( 1 9 8 5 ) . R e p l i c a ti o n a n d s u p e rc o i l i n g o f S i m i a n v i ru s 4 0 D N A
in ce ll e x tra c ts f ro m h u m a n c e l l s .
Wo
bbe, C . R .,
c i rc u l a r D N A
5710-5714.
Dea
n
,
F .,
Molec. cell. Biol. 5 ,
W e is s b a c h ,
L . and
2051-2060.
H u r w it z ,
c o n ta i n i n g th e s i m i a n v i ru s 4 0 D N A
In vitro r e p l i c a t i o n o f d u p l e x
Proc. natn. Acad. Sci. U.SA. 8 2 ,
J. ( 1 9 8 5 ) .
o ri g i n s i te .
Julian Blow