The binding of lamin B receptor to chromatin is regulatedby phosphorylation in the RS region Makoto Takano1, Masaki Takeuchi1, Hiromi Ito2, Kazuhiro Furukawa1,2,3, Kenji Sugimoto4, Sabur
Trang 1The binding of lamin B receptor to chromatin is regulated
by phosphorylation in the RS region
Makoto Takano1, Masaki Takeuchi1, Hiromi Ito2, Kazuhiro Furukawa1,2,3, Kenji Sugimoto4,
Saburo Omata1,2,3and Tsuneyoshi Horigome1,5
1
Courses of Biosphere Science and2Functional Biology, Graduate School of Science and Technology, Niigata University, Japan;
3
Department of Biochemistry, Faculty of Science, Niigata University, Japan;4Laboratory of Applied Molecular Biology, Department
of Applied Biochemistry, University of Osaka Prefecture, Osaka, Japan;5Center for Instrumental Analysis, Niigata University, Japan
Binding of lamin B receptor (LBR) to chromatin was studied
by means of an in vitro assay system involving recombinant
fragments of human LBR and Xenopus sperm chromatin
Glutathione-S-transferase (GST)-fused proteins including
LBR fragments containing the N-terminal region (residues
1–53) and arginine-serine repeat-containing region (residues
54–89) bound to chromatin The binding of GST-fusion
proteins incorporating the N-terminal and arginine-serine
repeat-containing regions to chromatin was suppressed by
mild trypsinization of the chromatin and by pretreatment
with a DNA solution A new cell-free system for analyzing
the cell cycle-dependent binding of a protein to chromatin
was developed from recombinant proteins, a Xenopus egg
cytosol fraction and sperm chromatin The system was
applied to analyse the binding of LBR to chromatin It was shown that the binding of LBR fragments to chromatin was stimulated by phosphorylation in the arginine-serine repeat-containing region by a protein kinase(s) in a synthetic phase egg cytosol However, the binding of LBR fragments was suppressed by phosphorylation at different residues in the same region by a kinase(s) in a mitotic phase cytosol These results suggested that the cell cycle-dependent binding of LBR to chromatin is regulated by phosphorylation in the arginine-serine repeat-containing region by multiple kinases Keywords: chromatin binding; lamin B receptor; LBR; Xenopusegg extract
The mature eggs of most vertebrates stay at the metaphase
of the second meiotic division until they meet sperm In that
phase, the nuclear envelope is dispersed in the cytoplasm as
nuclear envelope precursor vesicles Cell cycle progression is
triggered by fertilization, the nuclear envelope of the
pronucleus being formed first Then, the formation and
disruption of nuclear envelopes occurs repeatedly during
cleavage and in further differentiated somatic cell divisions
Thus, the structure of nuclear envelopes changes very
dynamically depending on the stage of the cell cycle To
ensure the precise assembly/disassembly of nuclear
envel-opes in the cell cycle, the binding of proteins on nuclear
envelope precursor vesicles/inner nuclear membranes to
chromatin should be precisely regulated
Major nuclear envelope proteins known to bind to
chromatin are lamins [1–4], lamin B receptor (LBR) [5,6],
and LAP2b [7,8] A peripheral nuclear membrane protein,
Ya, is also known as a chromatin binding protein in early
embryos of Drosophila melanogaster [9] LAP2 was found as lamina-associated polypeptides in rat liver nuclear envelopes and shown to bind to chromatin at the N-terminal region [7,8] It was shown recently that when a recombinant fragment of the protein was added to cell-free Xenopus egg nuclear assembly reactions at high concentrations, mem-brane binding to chromatin is inhibited [10] LBR was found first as an avian erythrocyte- and liver-nuclear membrane protein [11,12] Then, LBR was shown to be a chromatin-binding protein [5,6,13] The segment two-thirds from the C-terminal of the LBR molecule contains eight transmembrane-segments [6,14,15] and exhibits sterol C14 reductase activity [16,17] The segment one-third from the N-terminal (1–208) of human LBR is located in the nucleoplasm [14], and this portion is responsible for the binding of chromatin, DNA and most other proteins reported previously In chicken erythrocytes, an 18-kDa membrane protein [18] and an LBR kinase were found to be associated with LBR [19] LBR also bound a nuclear localization signal peptide [6,20], nucleoplasmin [6,20], and DNA [15] in vitro It was shown by means of a two hybrid method that heterochromatin protein 1 (HP1) binds to LBR [21], and the binding site was localized to a region (residues 97–174) of the N-terminal portion of human LBR [22] Importantly, it was shown that LBR, but not LAP2, is essential for the vesicle binding to chromatin using vesicles selectively depleted of these proteins by means of specific antibodies [5]
There have been some reports on regulation of the binding of LBR to other proteins Phosphorylation of the arginine-serine repeat-region in the N-terminal portion of LBR by an LBR kinase inhibits the binding of p34 protein
Correspondence to T Horigome, Department of Biochemistry,
Faculty of Science, Niigata University, 2-Igarashi, Niigata 950-2181,
Japan, Fax/Tel.: + 81 25 262 6160;
E-mail: thori@chem.sc.niigata-u.ac.jp
Abbreviations: CBB, Coomassie Brilliant Blue R-250; GST,
glutathi-one-S-transferase; LBR, lamin B receptor; HP1,
heterochromatin-associated protein 1; LAPs, lamina-heterochromatin-associated polypeptides;
PKA, protein kinase A; PKI, protein kinase inhibitor, a proteinous
inhibitor specific for protein kinase A; PKII, calmodulin-dependent
protein kinase II; SRPK, SR protein-specific kinase.
(Received 9 October 2001, revised 4 December 2001, accepted
7 December 2001)
Trang 2[23] LBR was phosphorylated in the mitotic phase in vivo
by an SR protein-specific kinase (SRPK) and cdc2 kinase
[24] However, phosphorylation of LBR by cdc2 kinase
in vitrohas no effect on the binding to lamin B [24] There
has been no report about the effect of phosphorylation on
the interaction of LBR and chromatin Therefore, a study
on the cell cycle-dependent regulation of the interaction of
LBR and chromatin is important for elucidation of the
nuclear envelope assembly/disassembly mechanism
In this study, we first determined the chromatin binding
region of LBR by using beads bearing recombinant
fragments of LBR and Xenopus sperm chromatin Then, a
system for analyzing the regulation mechanism for the
binding of LBR to chromatin was developed through the
combination of the above binding method and Xenopus egg
cytosol fractions It was suggested, with this method, that
the cell cycle-dependent binding of LBR to chromatin is
regulated by phosphorylation in the arginine-serine
repeat-containing region (RS-region) by multiple kinases The
potential function of LBR and LAP2 in vesicle targeting to
chromatin is discussed
M A T E R I A L S A N D M E T H O D S
Materials
Protein kinase inhibitors: A3 and K-252b were purchased
from Calbiochem Apyrase, the catalytic subunit of protein
kinase A, and protein kinase inhibitor (PKI) were obtained
from Sigma Chemicals Co Calmodulin-dependent protein
kinase II (PKII) was purified from bovine brain [25]
Buffers
NaCl/Pi: 10 mMsodium phosphate (pH 7.4), 140 mMNaCl
and 2.7 mM KCl; extraction buffer: 50 mM Hepes-KOH
(pH 7.7), 250 mMsucrose, 50 mMKCl and 2.5 mMMgCl2;
elution buffer: 25 mM Tris/HCl (pH 7.5), 150 mM NaCl
and 50 mM glutathione (reduced form); buffer X: 15 mM
Pipes-KOH (pH 7.4), 200 mM sucrose, 7 mM MgCl2,
80 mM KCl, 15 mM NaCl and 5 mM EDTA; SRPK
reaction buffer: 25 mM Tris/HCl (pH 7.5), 10 mM MgCl2
and 200 mM NaCl; and buffer M: 20 mM Hepes-KOH
(pH 7.5), 60 mM b-glycerophosphate, 20 mM EGTA and
15 mMMgCl2
Preparation of demembranatedXenopus sperm
chromatin
Xenopussperm was treated with lysolecithin to remove the
plasma and nuclear membranes without the highly
con-densed chromatin being affected, according to the method
of Smythe & Newport [26] The chromatin concentration is
expressed as the number of chromatin complexes in the
binding reaction mixture The number was determined by
counting with a hemacytometer
Preparation of a synthetic phaseXenopus egg cytosol
Xenopus eggs were collected, dejelled, and then lysed to
prepare a synthetic phase (interphase) extract, essentially as
described previously [27] The extraction buffer was
supple-mented with 2 m 2-mercaptoethanol, 10 lgÆmL)1
aproti-nin and leupeptin immediately before use Eggs were packed into tubes by brief centrifugation for several seconds at
6000 g Excess buffer above the packed eggs was removed and the eggs were then crushed by centrifugation at
15 000 g for 10 min The crude extract, i.e the supernatant between the lipid cap and pellet, was collected and mixed with 10 lgÆmL)1 cytochalasin B The crude extract was further separated into cytosol, membrane and gelatinous pellet fractions by ultracentrifugation at 200 000 g for 4 h in
an RP55S rotor (Hitachi, Tokyo) The cytosol fraction was then re-centrifuged at 200 000 g for 30 min to remove residual membranes and stored at)80 °C until use Preparation of a mitotic phaseXenopus egg extract Eggs were dejelled with 2% cysteine/NaOH (pH 8.0) at
23°C After washing three times with 100 mMNaCl and twice with buffer M at 23°C, the eggs were washed twice with cold buffer M containing 100 mMNaCl and 250 mM sucrose at 4°C Then the eggs were supplemented with
10 lgÆmL)1aprotinin and leupeptin, and packed into tubes
by brief centrifugation for several seconds at 6000 g Excess buffer above the packed eggs was removed and the eggs were then crushed by centrifugation at 15 000 g for 10 min The crude extract was collected, and further separated into cytosol, membrane and gelatinous pellet fractions as for the preparation of the synthetic phase extract, except that cytochalasin B was not added and buffer M was used instead of extraction buffer
Chromatin binding assay (I): a method involving soluble proteins
This method was used in the experiments for Fig 2
A cytosol fraction of Xenopus eggs was boiled for 10 min, cooled in ice-water for 5 min, and then centrifuged at
10 000 g for 10 min to remove denatured proteins The resulting supernatant, i.e heated cytosol, containing nucleo-plasmin was stored at )80 °C until use To determine chromatin binding of GST-fused proteins, 5 lL of demem-branated sperm chromatin (40 000 per lL) in buffer X was incubated with 50 lL of heated cytosol at 23°C for 30 min for decondensation of the chromatin Then the chromatin was precipitated by centrifugation at 2000 g for 10 min The pellet was suspended in 10 lL of extraction buffer contain-ing 0.1% Triton X-100 and 0.5 lg of GST, GST–NK, GST–
NM, GST–RS or GST–SK, and then incubated at 4°C for
20 min Chromatin was reprecipitated by centrifugation at
7000 g for 10 min The resulting supernatant was designated
as the Ôunbound fractionÕ The precipitated chromatin was washed with 200 lL of extraction buffer, and then dissolved
in 25 lL of 1% SDS The resulting solution was centrifuged
at 100 000 g for 1 h and the supernatant was designated as the Ôbound fractionÕ The obtained ÔboundÕ (20 lL) and ÔunboundÕ (8 lL) fractions were separated by SDS/PAGE, transferred to a nitrocellulose filter, and immunoblotted with affinity purified anti-GST Ig as described previously [28]
Chromatin binding assay (II): a method involving immobilized proteins
This method was used for all chromatin-binding experi-ments other than those in Fig 2 Demembranated sperm
Trang 3chromatin (40 000 per lL) in 0.5 lL of buffer X was
incubated with 10 lL of Xenopus egg heated cytosol at
23°C for 30 min for decondensation of the chromatin
Then the reaction mixture was centrifuged at 300 g for
10 min and the precipitated chromatin was suspended in
20 lL of extraction buffer After centrifugation, the
preci-pitated chromatin was resuspended in 10 lL of extraction
buffer, and then added to 2 lg of GST-fused proteins
attached to 2 lL of glutathione–Sepharose 4B beads
suspended in 10 lL of extraction buffer After incubation
at 4°C for 20 min, the binding reaction was stopped by
pipetting 10-lL samples onto glass slides spotted with 8 lL
of fixing solution (3% formaldehyde, 2 lLÆmL)1Hoechst
dye 33342, 80 mM KCl, 15 mM NaCl, 50% glycerol and
15 mMPipes, pH 7.2) The fixed samples were observed by
phase-contrast and fluorescence microscopy One hundred
beads were observed for every sample and ‘the percentage of
beads with bound chromatin’ was calculated This value
was used as an index of the affinity of beads bearing LBR
fragments and chromatin The values shown in the figures
are the averages of three or more experiments, and are
shown as values after subtraction of a blank value, except in
Fig 4 The blank value was determined in every experiment
using GST–Sepharose beads instead of GST–LBR
frag-ment-Sepharose beads, as shown in Fig 4 The bars in the
figure show the standard error
Assay for cell cycle dependency of the binding
of LBR fragments to sperm chromatin
GST-fused proteins attached to glutathione–Sepharose
beads were preincubated with either a synthetic or mitotic
phase egg cytosol fraction at 23°C for one hour After
washing twice with extraction buffer, the binding to
chromatin was examined by chromatin binding assay II,
as shown above The addition of 1M NaCl to the
washing buffer to remove possible bound proteins from
gel beads had no effect on the binding of chromatin to
beads
Expression of LBR fragments and preparation
of beads bearing these fragments
Cloning of various fragments of human LBR fused with
GST was carried out as previously described for
Escher-ichia coli[6] Expression of fusion proteins was induced by
the addition of 0.1 mM isopropyl thio-b-D-galactoside,
followed by incubation for 6 h at 30°C The bacterial
cells were collected by centrifugation and resuspended in a
buffered saline solution The cell suspension was sonicated
vigorously and then centrifuged at 15 000 g for 10 min
An aliquot of the prepared supernatant was reacted with
glutathione–Sepharose beads at 4°C for 2 h After
washing twice with the buffered saline solution, the beads
were stored at 4°C until use The amount of protein
immobilized on beads was estimated by the Lowry
method after elution with glutathione followed by
acetone-precipitation
Phosphopeptide mapping
GST–NK phosphorylated with [c-32P]ATP in vitro was
separated by SDS/PAGE and then transferred to a
nitrocellulose sheet The GST–NK band was excised, soaked in 0.5% poly(vinyl pyrrolidone) K)30 (Wako, Tokyo) in 100 mMacetic acid for 30 min at 37°C and then washed extensively with water The protein was digested with trypsin in 50 mMNH4HCO3at 37°C for 24 h The released peptides were dried, dissolved in water, and then loaded onto a cellulose TLC plate (Funacell; Funakoshi Co., Tokyo) Electrophoresis in the first dimension was performed at pH 8.9 (1% ammonium carbonate) for
20 min at 1000 V; ascending chromatography in the second dimension was performed using a solvent system of 37.5% 1-butanol, 25% pyridine and 7.5% acetic acid in water (v/v) The dried plate was exposed to Fuji X-ray film with intensifying screens
Preparation of a heterochromatin-associated protein 1 (HP1) fragment
Recombinant GST-fused HC1 (83–191 amino acids), an human HP1HSa fragment containing the LBR binding domain (104–191 amino acids) [22], was expressed as a GST-fusion protein in E coli, as previously described [29], and then bound to glutathione–Sepharose beads The beads were treated with Factor Xa to hydrolyze the hinge region of GST and HC1 at 37°C for 3 h Then, the cleaved HC1 portion, which was recovered in the supernatant, was concentrated and used for the binding assay
R E S U L T S
Identification of chromatin binding regions of LBR
To analyze the binding of LBR to chromatin, we used the N-terminal portion of LBR, because this portion is respon-sible for the binding to chromatin [5,6] A fragment containing the whole N-terminal portion, NK, and its subfragments, shown in Fig 1A, were expressed in E coli
as GST fusion proteins (Fig 1B), and then bound to glutathione–Sepharose beads These beads were incubated with demembranated and decondensed sperm chromatin After fixation and staining of DNA with Hoechst 33342, the beads were observed by phase contrast and fluorescence microscopy (Fig 1C) Most GST–NK (Fig 1C) and GST–
RS (not shown) beads bound chromatin, however, GST beads only bound a little (Fig 1C) Then, we introduced Ôpercentage of beads with bound chromatinÕ as an index for estimating the affinity of protein fragment-bearing beads with chromatin One hundred beads were counted and the percentage of beads with bound chromatin was calculated GST–NK, GST–RS and GST bearing beads gave values of
65 ± 7, 60 ± 5 and 18 ± 5%, respectively These values clearly show that the RS moiety within the NK region of LBR exhibits affinity with chromatin Then, to confirm these results, we tried an established in vitro chromatin-binding assay involving soluble proteins GST–NK, GST–
NM, GST–RS, GST–SK and GST in a soluble state were incubated with chromatin The chromatin bound and unbound fractions were analyzed by immunoblotting (Fig 2) GST–NK and GST–RS were bound to chromatin, although GST–NM, GST–SK and the GST moiety alone were not bound (Fig 2) Furthermore, bindings of GST–
NK and GST–RS to chromatin were inhibited in the
Trang 4presence of free DNA (Fig 2) The inhibition with DNA is
consistent with that observed with an assay involving
GST-fusion protein bearing beads, as shown below From these
results, we concluded that Ôthe percentage of beads with
bound chromatinÕ obtained with GST-fusion
protein-bear-ing beads can be used as an index of the affinity of protein
fragments to chromatin
We then applied this bead method to characterize the binding of LBR to chromatin because it is faster and needs only a one-tenth amount of chromatin compared to the established chromatin binding assay GST–NK, GST–NM, GST–RS and GST–SK beads were prepared, and chro-matin binding was examined (Fig 3, empty columns) It is
Fig 2 Chromatin binding assay involving soluble GST-fusion proteins Approximately 0.5 lg of GST–NK, GST–NM, GST–RS, GST–SK or GST was incubated with various amounts of decondensed Xenopus sperm chromatin, as shown in the figure, and then centrifuged to separate the unbound fraction (supernatant) from chromatin The pellet was washed with extraction buffer, dissolved in 1% SDS and then ultracentrifuged to remove DNA The thus obtained supernatant was designated the Ôbound fractionÕ The ÔboundÕ and ÔunboundÕ frac-tions were separated by SDS/PAGE and then analyzed by immuno-blotting with anti-GST Ig In the case of Ô(+ DNA)Õ, GST-fusion proteins were preincubated with 0.5 mgÆmL)1of porcine liver DNA and then used for the binding assay ÔUÕ and ÔBÕ in the figure denote the ÔunboundÕ and ÔboundÕ fractions, respectively For other details, see Materials and methods.
Fig 1 N-Terminal fragments of LBR expressed as GST fusion pro-teins, and the binding of beads bearing these fragments to chromatin (A) Schematic diagrams of N-terminal fragments of LBR expressed as GST fusion proteins The line numbered 1 and 211 shows the N-terminal portion of the LBR molecule, and these numbers are those
of amino-acid residues from the N-terminal of LBR ÔRSÕ is the site of arginine-serine repeats (B) SDS/PAGE of GST fusion proteins Samples were expressed in E coli, purified with glutathione–Sepha-rose, analyzed by SDS/PAGE on a 10% gel, and then stained with Coomassie Brilliant Blue R-250 (CBB) The lines at the left show the positions of marker proteins having relative molecular masses of 66, 43 and 29 kDa, from top to bottom (C) Binding of GST–NK bearing beads to chromatin GST and GST–NK bearing glutathione-Sepha-rose beads were incubated with decondensed Xenopus sperm chro-matin at 4 °C for 20 min, and then observed by phase contrast and fluorescence microscopy after staining of DNA with Hoechst 33342 Arrows, arrowheads and double-arrow heads indicate beads, unbound chromatin and bound chromatin, respectively Bar ¼ 10 lm.
Trang 5known that GST–NM, GST–RS and GST–SK carry
binding sites for chromatin [6], naked DNA [15], and a
heterochromatin specific protein, HP1 [22], respectively
Beads bearing GST–NK and GST–RS bound chromatin
GST–NM beads also showed apparent but lower affinity to
chromatin The lower affinity could not be detected in an
assay involving soluble proteins (Fig 2) These results
showed that the NM and RS regions have affinity for
chromatin However, the binding of chromatin to GST–SK
beads was very low, although the fragment in question
carries a binding site for chromatin protein HP1 This point
is discussed below
To characterize the mode of binding of LBR to
chromatin, beads bearing LBR fragments were
preincu-bated with a DNA solution and then the binding to
chromatin was examined (Fig 3, hatched columns) The
binding of RS- and NK-fragments to chromatin was
strongly suppressed, but the binding of the NM-fragment
was little affected These results suggested that the
RS region of LBR binds to the DNA region of
chromatin
On the other hand, with the pretreatment of the
chromatin with a low concentration of trypsin, the
binding was suppressed strongly in the case of the NM
fragment but not the NK and RS fragments (Fig 3, filled
columns) These results suggested that the NM region
binds to a protein(s) on chromatin These results also
suggested that the binding of the RS region to chromatin
DNA is superior to the binding of the NM region to the
chromatin protein because the binding mode of NK,
which contains the NM and RS regions, is similar to that
of RS
An assay system for the cell cycle-dependent binding
of LBR to chromatin
We wondered whether this binding system can be applied to the analysis of the cell cycle-dependent interaction of LBR and chromatin Therefore, we pretreated beads bearing LBR fragments with a Xenopus egg cytosol fraction at the synthetic phase of the cell cycle, and then chromatin binding was examined The binding was stimulated (data not shown) When NK beads were pretreated with a mitotic phase cytosol fraction, however, the binding was strongly suppressed (data not shown) Changes in the affinity of chromatin to NK on pretreatment with the two phases egg extracts were the same as the predicted changes in living cells These preliminary results suggested that an in vitro assay system for the analysis of the cell cycle-dependent interaction of LBR and chromatin can be developed using this binding assay system
Then, we optimized the assay conditions for analysis of the cell cycle-dependent interaction of LBR and chro-matin (Fig 4) The preincubation time for GST–NK beads at 23 °C with a synthetic phase cytosol fraction was examined and it was found that 60 min is necessary
to reach a plateau of increased binding affinity (Fig 4A) The same preincubation time was applicable to experi-ments involving a mitotic phase cytosol fraction (data not shown) The binding of chromatin to GST–NK beads almost linearly increased with increasing chromatin con-centration up to 70–80% (Fig 4B) Various concentra-tions of GST–NK on beads, 1–10 lgÆlL)1, had no effect
on the percentage of beads with bound chromatin (data not shown) The binding of chromatin to GST–NK beads was very fast, being completed within one minute at 4°C (Fig 4C) Then, as standard conditions, we chose 60 min
as the preincubation time, 20 000 chromatin per assay, and 20 min for the time of binding of chromatin to beads, as shown under Materials and methods The chromatin concentration can be varied, depending on the experimental purpose, i.e lower and higher chromatin concentrations can be used to analyze increases or decreases in binding activity (for example, Fig 5A,B) Then, we applied this method to analyze the regulation mechanism for the binding of LBR to chromatin, as described below
Cell cycle-dependent binding of LBR fragments
to chromatin When beads were pretreated with a synthetic phase cytosol fraction, the numbers of NK- and RS-beads with bound chromatin were significantly increased, but not that
of NM-beads (Fig 5A) SK-beads showed no significant binding of chromatin regardless of treatment with a synthetic phase cytosol fraction (Fig 5A) On the other hand, when beads were pretreated with a mitotic phase cytosol fraction, the numbers of NK- and RS-beads with bound chromatin were significantly decreased, but not that of NM-beads (Fig 5B) SK-beads again showed no significant binding regardless of treatment with a mitotic phase cytosol fraction (Fig 5B) These results show that the affinity of the N-terminal portion of LBR and chromatin increases in a synthetic phase extract and decreases in a mitotic phase one in vitro Moreover, the
Fig 3 Identification of chromatin binding domains in the N-terminal
region of LBR and analysis of the binding mode Empty columns: four
kinds of GST fusion proteins including N-terminal domains of LBR
attached to glutathione–Sepharose beads were incubated with
decon-densed sperm chromatin at 4 °C for 20 min, and then observed by
fluorescence microscopy after staining of DNA with Hoechst 33342.
The Ôpercentage of beads with bound chromatinÕ values were
deter-mined as described under Materials and methods after subtraction of
the value for blank GST-beads (Hatched columns) Four GST fusion
proteins including LBR fragments attached to glutathione–Sepharose
beads were preincubated with a 0.5-mgÆmL)1DNA solution at 4 °C
for 1 h, and then the binding to chromatin was examined as above.
(Filled columns) Decondensed chromatin was pretreated with
10 lgÆmL)1trypsin at 23 °C for 10 min, and then after the addition of
leupeptin and aprotinin (final, 0.5 mgÆmL)1), the binding to beads
bearing GST–LBR fragments was examined as above.
Trang 6binding of the N-terminal portion of LBR to chromatin
is regulated through the RS region, not the NM- and
SK-regions The directions of the changes in the affinity
of NK and chromatin on treatment with the two
cytosol fractions in vitro, i.e., increasing with a synthetic
phase cytosol fraction decreasing with a mitotic one, were
strikingly the same as those of the changes in the affinity
of nuclear envelope precursor vesicles and chromatin
in vivo The results obtained with this in vitro system
seemed to reflect this phenomenon in vivo
Stimulation of the binding of LBR to chromatin
by phosphorylation by a kinase(s) in a synthetic phase egg cytosol
Chromatin binding of beads bearing GST–NK was incr-eased by pretreatment with a synthetic phase egg cytosol fraction (compare columns 1 and 2 in Fig 6A) The increase could be suppressed by apyrase and protein kinase-inhib-itors having broad specificities: staurosporine, A3 [30], and K252b (compare columns 3–6 with column 2 in Fig 6A) Fifty percent suppression with staurosporine was achieved with as little as 4 nM (data not shown) These results indicate that the increase in the affinity of NK to chromatin
is an ATP-dependent reaction, and is caused by a kinase(s)
in the cytosol Then, authentic protein kinase A (PKA) and calmodulin-dependent protein kinase II (CaMKII) were applied instead of the cytosol fraction, as we previously observed that LBR is phosphorylated by these kinases [28] PKA but not PKII caused a similar increase in the affinity
of NK to chromatin (Fig 6A, column 7) However, protein kinase inhibitor (PKI), a PKA-specific inhibitor, could not suppress the stimulation of the binding of NK to chromatin
Fig 5 Effects of pretreatment of LBR fragments with Xenopus egg cytosol fractions on the binding to chromatin (A) Beads, with bound GST and GST-fused proteins including LBR fragments, were pre-treated with extraction buffer (empty bars) or a synthetic phase egg cytosol fraction (filled bars), and then the binding to chromatin was determined as in Fig 3 (B) The same as (A) except that a mitotic phase egg cytosol fraction was used instead of the synthetic phase one Instead of 20 000 chromatin per assay as a standard condition, 10 000 and 25 000 chromatin per assay were used in (A) and (B), respectively,
to clearly show the changes in Ôpercentage of beads with bound chromatinÕ.
Fig 4 Assay conditions for the binding of chromatin to GST–NK beads
pretreated with a synthetic phase Xenopus egg cytosol fraction GST–
NK (filled circles) and blank GST (open circles) beads, 2 lL, were
preincubated with 20 lL of a synthetic phase Xenopus egg cytosol
fraction at 23 °C for 1 h Thus treated beads were then incubated with
25 000 chromatin per assay at 4 °C for 20 min Then, the percentage of
beads with bound chromatin was determined In each figure, the time
of preincubation of beads with a cytosol fraction (A), the chromatin
concentration (B), or the time of incubation of beads with chromatin
(C) was varied.
Trang 7by a synthetic phase cytosol (Fig 6A, column 9) These
results suggested that a kinase(s) in the synthetic phase egg
cytosol fraction phosphorylates NK at a functionally similar
site(s) to in the case of PKA, and thereby increases the
affinity of LBR and chromatin The binding of chromatin
to GST–RS beads was also stimulated by a synthetic phase
cytosol and PKA (Fig 6A, columns 10–13) These data
suggested that phosphorylation at a site(s) within the RS
region is responsible for the stimulation To confirm the
phosphorylation in the RS region, beads bearing GST, GST–NK and GST–RS were incubated with a synthetic phase cytosol in the presence of [c-32P]ATP Then, the proteins were eluted with SDS and analyzed by SDS/ PAGE The gel was stained with CBB and then subjected to autoradiography (Fig 6B) Radioactivity was detected for the GST–NK and GST–RS beads, but not for GST itself (Fig 6B, autoradiography, lanes 1–3) Incorporation of radioactivity into the GST–NK and GST–RS bands was completely suppressed by the addition of staurosporine (Fig 6B, autoradiography, lanes 5 and 6) These results indicate that LBR is indeed phosphorylated in the RS region by a synthetic phase cytosol, which stimulated the binding to chromatin
Suppression of the binding of LBR to chromatin
by phosphorylation with a kinase(s) in a mitotic phase egg cytosol
The affinity of NK-beads and chromatin was decreased by preincubation of the beads with a mitotic phase egg cytosol fraction (compare columns 1 and 2 in Fig 7A) The decrease could be suppressed by apyrase and protein kinase inhibitors having broad specificities: staurosporine, A3 and K252b (Fig 7A) Fifty percent suppression with stauro-sporine was achieved with 6 nM(data not shown) These results suggest that the decrease in the affinity of NK and chromatin is an ATP-dependent reaction, and is caused by a kinase(s) in the cytosol When GST–RS was used instead of GST–NK, similar suppression of the binding to chromatin was observed with preincubation with a mitotic cytosol (Fig 7A, columns 7–9) These results suggested that phos-phorylation in the RS region is responsible for the suppression To confirm the phosphorylation in the RS region, beads bearing GST–NK, GST–RS and only GST were incubated with a mitotic phase cytosol fraction in the presence of [c-32P]ATP Then, the proteins were eluted with SDS and analyzed by SDS/PAGE, followed by CBB staining and autoradiography (Fig 7B) Radioactivity was detected for the GST–NK and GST–RS beads, but not for GST (Fig 7B, Autoradiography, lanes 1–3) Incorporation
of the radioactivity into the GST–NK and GST–RS bands was completely suppressed by the addition of staurosporine (Fig 7B, autoradiography, lanes 5 and 6) These results indicate that LBR is phosphorylated in the RS region by a mitotic phase cytosol, which suppressed the binding to chromatin
Phosphopeptide mapping Synthetic phase and mitotic phase egg extracts both phosphorylated GST–NK and had opposite effects on chromatin binding affinity (Figs 6 and 7) Therefore, the phosphorylation sites for the two extracts were expected to
be different Then, to confirm this difference, tryptic phosphopeptides of GST–NK treated with synthetic phase and mitotic phase egg extracts were compared with each other by means of two-dimensional separation (Fig 8) As can be seen in Fig 8, several phosphopeptide spots were different, although some were the same These results clearly showed that the NK fragment is phosphorylated with synthetic phase and mitotic phase egg extracts at common multiple sites, however, as expected, several sites are
Fig 6 Stimulation of the binding of LBR fragments to chromatin by
phosphorylation with a synthetic phase egg cytosol fraction (A) Effects
of apyrase, protein kinases, and protein kinase inhibitors on
stimula-tion of the binding of LBR fragments to chromatin by pretreatment
with a synthetic phase egg cytosol fraction GST–NK-beads (columns
1–9) and GST–RS-beads (columns 10–13) were preincubated with
extraction buffer (Buffer), a synthetic phase egg cytosol fraction (SC),
1 lgÆmL)1protein kinase A (PKA), 1 lgÆmL)1calmodulin-dependent
protein kinase II (CaMKII), and SC containing either 8 mU apyrase,
10 n M staurosporine (Sta.), 1 m M A3, 1 l M K252b or 50 lgÆmL)1
protein kinase inhibitor (PKI), and then the binding to chromatin was
examined as in Fig 3 (B) Detection of phosphorylation Beads
bearing 1–2 lg GST, GST–NK, or GST–RS were incubated with
20 lL of a synthetic phase egg cytosol fraction supplemented with
0.1 lL of 3.3 l M [c- 32 P]ATP (110 TBqÆmmol)1) in the presence
(SC/Sta.) or absence (SC) of 10 l M staurosporine at 23 °C for 1 h.
Thus treated proteins were extracted with SDS and then analyzed by
SDS/PAGE, followed by CBB staining and autoradiography Lanes 1
and 4, GST; lanes 2 and 5, GST–NK; lanes 3 and 6, GST–RS.
The arrowhead and double arrowhead indicate the GST–NK and
GST–RS bands, respectively.
Trang 8phosphorylated specifically with a synthetic phase or mitotic phase extract
D I S C U S S I O N
Binding sites on the N-terminal portion of LBR for chromatin
Ye et al reported that free-DNA [15] and a chromatin protein, HP1 [22], bind with LBR in regions corresponding
to the RS and SK regions, respectively On the other hand,
we previously reported that the NM region of LBR, which is different from the RS and SK regions, binds with chromatin [6] Therefore, we analyzed the binding of LBR to chromatin
in more detail using an assay method involving GST-fusion fragments of LBR and Xenopus sperm chromatin in this study It was shown that the RS region of LBR binds with chromatin and that the binding is inhibited by the addition
of free DNA (Figs 2 and 3) These results suggested that LBR binds to a DNA region on chromatin in the RS region This idea was consistent with a report by Ye & Worman [15], i.e that a region corresponding to RS binds free DNA Duband-Goulet & Courvalin recently showed that LBR binds linker DNA but not the nucleosome core using in vitro reconstituted nucleosomes and short DNA fragments [31] Therefore, the binding site on chromatin for the RS region
of LBR seems to be linker DNA
On the other hand, it was suggested that the NM-region, not the SK-region, binds to a protein(s) on sperm chromatin ([6]; Fig 3) HP1, the only known chromatin protein which binds to LBR, was reported by Ye et al to bind to a region
of SK [22] Then, we examined which region of LBR binds
to HP1 in our binding assay system A HP1 fragment (83–191 amino acids) containing the LBR binding region was expressed in E coli, and then the binding to beads bearing GST–NK, GST–NM, GST–RS and GST–SK was examined The HP1 fragment bound to beads bearing GST–NK and GST–SK, but not to ones bearing GST–NM (data not shown) These results are consistent with those reported by Ye et al [22] On the other hand, James et al reported that HP1 is not observed in the nuclei of early syncytial embryos, but becomes concentrated in the nuclei
at the syncytial blastoderm stage (about nuclear division cycle 10) in Drosophila melanogaster [32] Therefore, HP1 may not participate in the binding of LBR to sperm chromatin in eggs In the case of the binding of LBR to
Fig 7 Suppression of the binding of LBR fragments to chromatin by
phosphorylation with a mitotic phase egg cytosol fraction (A) Effects of
apyrase and protein kinase inhibitors on suppression of the binding of
LBR fragments to chromatin by pretreatment with a mitotic phase egg
cytosol fraction GST-NK-beads (columns 1–6) and GST–RS-beads
(columns 7–9) were preincubated with extraction buffer (Buffer), a
mitotic phase egg cytosol fraction (MC), and MC containing either
8 mU apyrase, 10 n M staurosporine (Sta.), 1 m M A3 or 1 l M K252b,
and then the binding to chromatin was examined as in Fig 3.
(B) Beads bearing 1–2 lg GST, GST–NK, or GST–RS were treated
and analyzed as in Fig 6B, except that a mitotic phase egg cytosol
fraction was used instead of the synthetic phase one Lanes 1 and 4,
GST; lanes 2 and 5, GST–NK; lanes 3 and 6, GST–RS The arrowhead
and double arrowhead indicate the GST–NK and GST–RS bands,
respectively.
Fig 8 Tryptic phosphopeptide analysis of GST-NK Beads bearing 20 lg GST–NK were incubated with 20 lL of a synthetic phase (SC) or a mitotic phase (MC) egg cytosol fraction supplemented with 2 lL of 3.3 l M [c-32P]ATP (110 TBqÆmmol)1) at 23 °C for 1 h The thus treated proteins were separated by SDS/PAGE and then transferred to a nitrocellulose sheet The GST–NK bands were excised and digested with trypsin The eluted phosphopeptides were separated by electrophoresis at pH 8.9 (horizontal direction; cathode to the left) and by ascending chromato-graphy The points of sample application can be seen as dots near the bottom-left corners.
Trang 9sperm chromatin in eggs, binding through the RS region of
LBR to linker DNA of chromatin seems to be predominant
Then, the binding is supported by the interaction of the NM
region and a protein(s) other than HP1 on sperm chromatin
An assay system for the cell cycle-dependent binding
of LBR to chromatin
To analyze the regulatory mechanism for the cell
cycle-dependent binding of chromatin and nuclear membranes,
we developed a new in vitro assay system comprising a
Xenopusegg extract and a binding assay involving sperm
chromatin and beads bearing LBR fragments The binding
was stimulated by preincubation of beads bearing LBR
fragments with a synthetic phase extract, whereas it was
suppressed by that with a mitotic phase extract (Fig 5) The
binding of chromatin to LBR fragments on beads could be
estimated semiquantitatively by this method The effects of
enzymes, inhibitors and other reagents on the cell cycle–
dependent interaction could also be examined very easily by
means of this method This method is applicable to the
analysis of phosphorylated residues on LBR fragments and
protein kinases responsible for the phosphorylation This
method should also be applicable to the analysis of cell
cycle-dependent regulation of the binding of other proteins
to chromatin, such as LAP2, emerin, MAN1, lamins and
nuclear matrix proteins
Kinases responsible for cell cycle-dependent regulation
of the binding of LBR to chromatin
It was suggested that the binding of LBR to sperm
chromatin is stimulated by phosphorylation in the RS
region of LBR by a kinase(s) in a synthetic phase egg cytosol
(Fig 6) In interphase somatic cell nuclei, the binding of
LBR to lamin B may be stimulated by in vivo
phosphory-lation by PKA [33] However, PKA seems not to participate
in stimulation of the binding of LBR to chromatin in a
Xenopusegg system, because PKI, a specific inhibitor for
PKA, could not suppress the stimulation (Fig 6A) On the
other hand, an SR-repeat specific kinase 1 (SRPK1), which
is expressed ubiquitously [34] and phosphorylates LBR in a
constitutive manner, is known to phosphorylate serine/
threonine residues within the RS-repeat (Fig 1) in the RS
region at multiple sites [23,35] This phosphorylation
inhibits the binding of LBR to p34/p32 [23], whereas there
has been no report about the effect on the interaction of
LBR and chromatin Identification of the kinase(s)
respon-sible for the stimulation of the binding of LBR to chromatin
is important for clarifying the physiological function of the
phosphorylation, and such work is currently underway
It was suggested that the binding of LBR to sperm
chromatin is strongly suppressed by phosphorylation in the
RS region of LBR by a kinase(s) in a mitotic phase egg
cytosol (Fig 7) Results of phosphopeptide mapping of
GST–NK treated with synthetic phase and mitotic phase
egg extracts showed different patterns (Fig 8) It is known
that recombinant cdc2 kinase and a mitotic extract of
cultured chicken cells phosphorylate serine 71 within the RS
region [24] The binding of LBR to lamin B is not affected
by such phosphorylation, whereas the effect on the binding
to chromatin is not known [24] In a zebrafish egg system, it
was found that PKC and cdc2-kinase mediate
phosphory-lation events that elicit nuclear envelope disassembly [36]
In a sea urchin egg system, an LBR-like protein mediates targeting of nuclear envelope vesicles to sperm chromatin [37] These observations are consistent with the idea that phosphorylation of serine 71 of LBR by cdc2 kinase in a mitotic egg cytosol participates in the dissociation of LBR and chromatin Therefore, identification of the kinase(s) and phosphorylation site(s) responsible for the suppression
is important, and such work is currently underway
Cell cycle-dependent regulation of the interaction
of nuclear membranes and chromatin The dissociation/association of membranes with chromatin
in pronuclei formation, and the beginning and end of mitosis are critical for control of the nuclear dynamics during these stages of the cell cycle Inner nuclear membrane proteins, LBR [5,6,13], LAP2 [7], and emerin (M Segawa,
K Furakawa, S Omata & T Horigone, unpublished observations) have been shown to bind directly to chrom-atin Therefore, precise regulation of the cell-cycle depen-dent dissociation/association of these proteins and chromatin is important for the cell cycle In the case of LBR, binding to chromatin was shown by sperm chromatin
in vitro([6,13]; this study), and by mitotic phase chromo-somes from CHO cells [5] Regulation of the binding of LBR to chromatin by phosphorylation was shown in this study using sperm chromatin and a Xenopus egg extract
In the case of LAP2, phosphorylation in the interphase [38] and mitotic phase [7] has been shown in somatic cells It has also been shown that the phosphorylation of LAP2 with a mitotic HeLa cell extract inhibits its binding to chromo-somes [7] In the cases of emerin and MAN1, which share the LEM domain with LAP2 [39,40], the regulatory mechanism for the binding to chromatin remains to be elucidated
The function of LBR in the targeting of nuclear membranes or nuclear envelope precursor vesicles to chromatin remains elusive In rat liver and turkey erythro-cyte in vitro systems, Pyrpasopoulou et al [5] showed that the binding of vesicles to chromatin was suppressed when LBR, but not LAP2, was immuno-depleted from the vesicles In a Xenopus egg cell-free system, however, it was found that vesicles containing NEP-B78 bind first to chromatin and then to vesicles containing an LBR-like protein [41] LBR-containing vesicles alone can not bind to chromatin [41] These observations may suggest that LBR does not participate in the binding of vesicles However, surface remodeling of chromatin through initial interactions between NEP-B78 containing vesicles and chromatin may permit LBR-chromatin binding activity [41] Therefore, the possibility of direct participation of LBR in cell cycle-dependent vesicle targeting to chromatin still remains for the Xenopusegg system, too In the case of the sea urchin egg system, it was suggested that a 56-kDa LBR-like protein, which reacts with anti-(human LBR) Ig, participates in the targeting [37] Therefore, the participation of LBR in the targeting of nuclear membranes to chromatin may vary a little from system to system and/or LBR acts together with other proteins Indeed, LBR and a LEM domain protein, emerin, are targeted to different regions on the surface of chromatin in the telophase very early, suggesting that the two proteins may participate in the targeting of nuclear
Trang 10membranes to different regions on the surface of chromatin
[42] We also observed the binding of a truncated emerin
protein directly to sperm chromatin in vitro (M Segawa,
K Furakawa, S Omata & T Horigone, unpublished
observation) LAP2 seems to participate in the targeting of
nuclear envelope precursor vesicles in the Xenopus egg
extract system because membrane binding to chromatin is
inhibited on the addition of a high concentration of a
truncated recombinant LAP2 protein to the cell-free
Xenopus egg extract system [10] Further analysis of the
regulation mechanism for the binding of a set of inner
nuclear membrane proteins to chromatin is necessary for
understanding the molecular mechanism of dissociation/
association of membranes with chromatin in pronucleus
formation and the mitotic phase of somatic cells
A C K N O W L E D G E M E N T S
We wish to thank Dr Masatoshi Hagiwara for the helpful discussion.
We also wish to thank Hitomi Susa and Satomi Hoshino for their help
in the construction of the plasmids encoding GST–RS and the
phosphopeptide mapping, respectively.
This work was supported by a Grant-in-Aid from the Ministry of
Education, Science, Sports and Culture of Japan, and grants from the
Biodesign Research Project and for Project Research of Niigata
University.
R E F E R E N C E S
1 Glass, J.R & Gerace, L (1990) Lamins A and C bind and
assemble at the surface of mitotic chromosomes J Cell Biol 111,
1047–1057.
2 Taniura, H., Glass, C & Gerace, L (1995) A chromatin binding
site in the tail domain of nuclear lamins that interacts with core
histones J Cell Biol 131, 33–44.
3 Lourim, D & Krohne, G (1998) Chromatin binding and
poly-merization of the endogenous Xenopus egg lamins: the opposing
effects of glycogen and ATP J Cell Sci 111, 3675–3686.
4 Stuurman, N., Heins, S & Aebi, U (1998) Nuclear lamins: their
structure, assembly, and interactions J Struct Biol 122, 42–66.
5 Pyrpasopoulou, A., Meier, J., Maison, C., Simos, G & Georgatos,
S.D (1996) The lamin B receptor (LBR) provides essential
chromatin docking sites at the nuclear envelope EMBO J 15,
7108–7119.
6 Kawahire, S., Takeuchi, M., Gohshi, T., Sasagawa, S., Shimada,
M., Takahashi, M., Abe, T.K., Ueda, T., Kuwano, R., Hikawa,
A., Ichimura, T., Omata, S & Horigome, T (1997) cDNA cloning
of nuclear localization signal binding protein NBP60, a rat
homologue of lamin B receptor, and identification of binding sites
of human lamin B receptor for nuclear localization signals and
chromatin J Biochem 121, 881–889.
7 Foisner, R & Gerace, L (1993) Integral membrane proteins of the
nuclear envelope interact with lamins and chromosomes, and
binding is modulated by mitotic phosphorylation Cell 73, 1267–
1279.
8 Furukawa, K., Fritze, C.E & Gerace, L (1998) The major nuclear
envelope targeting domain of LAP2 coincides with its lamin
binding region but is distinct from its chromatin interaction
domain J Biol Chem 273, 4213–4219.
9 Lopez, J.M & Wolfner, M.F (1997) The developmentally
regu-lated Drosophila embryonic nuclear lamina protein ÔYoung ArrestÕ
(fs(1)Ya) is capable of associating with chromatin J Cell Sci 110,
643–651.
10 Gant, T.M., Harris, C.A & Wilson, K.L (1999) Roles of LAP2
proteins in nuclear assembly and DNA replication: truncated
LAP210 proteins alter lamina assembly, envelope formation, nuclear size, and DNA replication efficiency in Xenopus laevis extracts J Cell Biol 144, 1083–1096.
11 Worman, H.J., Yuan, J., Blobel, G & Georgatos, S.D (1988)
A lamin B receptor in the nuclear envelope Proc Natl Acad Sci USA 85, 8531–8534.
12 Bailer, S.M., Eppenberger, H.M., Griffiths, G & Nigg, E.A (1991) Characterization of a 54-kDa protein of the inner nuclear membrane: evidence for cell cycle–dependent interaction with the nuclear lamina J Cell Biol 114, 389–400.
13 Gajewski, A & Krohne, G (1999) Subcellular distribution of the Xenopus p58/lamin B receptor in oocytes and eggs J Cell Sci 112, 2583–2596.
14 Worman, H.J., Evans, C.D & Blobel, G (1990) The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains J Cell Biol.
111, 1535–1542.
15 Ye, Q & Worman, H.J (1994) Primary structure analysis, and lamin B and DNA binding of human LBR, an integral protein of the nuclear envelope inner membrane J Biol Chem 269, 11306– 11311.
16 Silve, S., Dupuy, P.-H., Ferrara, P & Loison, G (1998) Human lamin B receptor exhibits sterol C14-reductase activity in Saccharomyces cerevisiae Biochim Biophys Acta 1392, 233–244.
17 Prakash, A., Sengupta, K., Aparna, K & Kasbekar, D.P (1999) The erg-3 (sterol D14,15-reductase) gene of Neurospora crassa: generation of null mutants by repeat-induced point mutation and complementation by proteins chimeric for human lamin B recep-tor sequences Microbiology 145, 1443–1451.
18 Simos, G., Maison, C & Georgatos, S.D (1996) Characterization
of p18, a component of the lamin B receptor complex and a new integral membrane protein of the avian erythrocyte nuclear envelope J Biol Chem 271, 12617–12625.
19 Simos, G & Georgatos, S.D (1992) The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins EMBO J 11, 4027–4036.
20 Haino, M., Kawahire, S., Omata, S & Horigome, T (1993) Purification of a 60 kDa nuclear localization signal binding protein
in rat liver nuclear envelopes and characterization of its properties.
J Biochem 113, 308–313.
21 Ye, Q & Worman, H.J (1996) Interaction between an integral protein of the nuclear envelope inner membrane and human chromodomain proteins homologous to Drosophila HP1 J Biol Chem 271, 14653–14656.
22 Ye, Q., Callebaut, I., Pezhman, A., Courvalin, J.C & Worman, H.J (1997) Domain–specific interactions of human HP1-type chromodomain proteins and inner nuclear membrane protein LBR J Biol Chem 272, 14983–14989.
23 Nikolakaki, E., Simos, G., Georgatos, S.D & Giannakouros, T (1996) A nuclear envelope-associated kinase phosphorylates arginine-serine motifs and modulates interactions between the lamin B receptor and other nuclear proteins J Biol Chem 271, 8365–8372.
24 Nikolakaki, E., Meier, J., Simos, G., Georgatos, S.D & Gian-nakouros, T (1997) Mitotic phosphorylation of the lamin B receptor by a serine/arginine kinase and p34cdc2 J Biol Chem.
272, 6208–6213.
25 Yamauchi, T & Fujisawa, H (1983) Purification and character-ization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-mono-oxygenase Eur J Biochem 132, 15–21.
26 Smythe, C & Newport, J.W (1991) Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts Methods Cell Biol 35, 449–468.
27 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.