Oncogene (1997) 14, 1611 ± 1615
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
Phosphorylation of DNA polymerase a-primase by Cyclin A-dependent
kinases regulates initiation of DNA replication in vitro
C Voitenleitner1,2, E Fanning1,2, and H-P Nasheuer1,3
1
Institut fuÈr Biochemie, LMU MuÈnchen, WuÈrmtalstr. 221, 81375 MuÈnchen, Germany; 2Department of Molecular Biology,
Vanderbilt University, Nashville, Tennessee 37235, USA; 3Institut fuÈr Molekulare Biotechnologie, Abteilung Biochemie,
Beutenbergerstrasse 11, 07745 Jena, Germany
DNA polymerase a-primase is the only known eukaryotic enzyme that can start DNA replication de novo. In
this study, we investigated the regulation of DNA
replication by phosphorylation of DNA polymerase aprimase. The p180 and the p68 subunits of DNA
polymerase a-primase were phosphorylated using Cyclin
A-, B- and E- dependent kinases. This phosphorylation
did not in¯uence its DNA polymerase activity on
activated DNA, but slightly stimulated primase activity
using poly(dT) single-stranded DNA (ssDNA) without
changing the product length of primers. In contrast, sitespeci®c initiation of replication on plasmid DNA
containing the SV40 origin is a€ected: Cyclin A-Cdk2
and Cyclin A-Cdc2 inhibited initiation of SV40 DNA
replication in vitro, Cyclin B-Cdc2 had no e€ect and
Cyclin E-Cdk2 stimulated the initation reaction. DNA
polymerase a-primase that was pre-phosphorylated by
Cyclin A-Cdk2 was completely unable to initiate the
SV40 DNA replication in vitro; Cyclin B-Cdc2-phosphorylated enzyme was moderately inhibited, while
Cyclin E-Cdk2-treated DNA polymerase a-primase
remained fully active in the initiation reaction.
Keywords: DNA replication; cyclins; cyclin-dependent
kinases; DNA polymerase a-primase
Introduction
Replication of SV40 DNA in vitro serves as an
excellent model system to study eukaryotic DNA
replication and its regulation (Challberg and Kelly,
1989; D'Urso et al., 1990; Fanning, 1994; Hurwitz et
al., 1990; Li and Kelly, 1984; Stillman, 1989; Waga et
al., 1994). Recently, it was shown that ten proteins or
protein complexes are necessary and sucient for SV40
DNA replication in vitro (Hurwitz et al., 1990; Waga et
al., 1994) o€ering the possibility to analyse regulation
of DNA replication with puri®ed proteins only.
Moreover, biochemical studies of the initiation of
SV40 DNA replication allowed the development of
an experimental model for the initiation reaction
(Challberg and Kelly 1989; Fanning, 1994; Fanning
and Knippers, 1992; Hurwitz et al., 1990; Stillman,
1989, Wang, 1991). T antigen, RP-A, and DNA
polymerase a-primase, that are required for initiation
Correspondence: H-P Nasheuer
Received 15 February 1996; revised 22 November 1996; accepted 25
November 1996
of SV40 DNA replication, are potential substrates of
Cyclin-dependent kinases (Cdks; Dutta and Stillman
1992; McVey et al., 1989; Nasheuer et al., 1991, 1992).
Since RP-A and DNA polymerase a-primase are key
cellular replication proteins, and they are phosphorylated in a cell cycle-dependent manner, it was suggested
that their phosphorylation might be important for the
regulation of DNA replication (Din et al., 1990; Dutta
and Stillman 1992; Ferrari et al., 1996; Foiani et al.,
1995; Nasheuer et al., 1991).
To determine functional interactions of Cdks and
replication proteins, we used the in vitro SV40 DNA
replication system. Four Cyclin-Cdks, i.e. Cyclin ACdc2, Cyclin A-Cdk2, Cyclin B-Cdc2, and Cyclin ECdk2, phosphorylated DNA polymerase a-primase, but
only Cyclin A-Cdc2 and Cyclin A-Cdk2 prevented
initiation of SV40 DNA replication in vitro. In
contrast, Cyclin E-Cdk2 and Cyclin B-Cdc2 showed a
modest e€ect.
Results
Phosphorylation of DNA polymerase a-primase by
Cyclin-Cdk complexes
To study the function of protein phosphorylation on
DNA replication, we puri®ed highly active, recombinant DNA polymerase a-primase, Cyclin A-Cdc2,
Cyclin A-Cdk2, Cyclin B-Cdc2 and Cyclin E-Cdk2.
The catalytic subunits Cdc2 and Cdk2 phosphorylated
neither histone H1 nor DNA polymerase a-primase
above background level (Figure 1a; data not shown).
The puri®ed Cyclin-Cdk complexes have reproducibly
high kinase activity on histone H1 and on DNA
polymerase a-primase as a substrate, but they
phosphorylated exclusively the p180 and p68 subunits
(Figure 1a; data not shown). Similar activities (adjusted
with histone H1) of each kinase complex were used to
phosphorylate DNA polymerase a-primase. Normalizing kinase activity this way, Cyclin A-Cdk2 kinase
had the highest activity in phosphorylation of the p180
and p68 subunits of DNA polymerase a-primase;
Cyclin A-Cdc2, Cyclin B-Cdc2 and Cyclin E-Cdk2
complexes were slightly less active in p180 and p68
phosphorylation (Figure 1a).
DNA polymerase a-primase that was pre-incubated
with increasing amounts of kinase complexes showed
the same DNA polymerase activity on activated DNA
as the control enzyme, while its primase activity, that
was measured on poly(dT)-ssDNA, was stimulated up
Regulation of DNA replication by phosphorylation
C Voitenleitner et al
1612
Cyclin B-Cdc2
Cyclin E-Cdk2
Cdc2
Cdk2
control
—
—
—
—
—
Cyclin A-Cdk2
195
130
87
66
54
Cyclin A-Cdc2
a
1
2
3
4
5
6
7
— p180 —
— p68 —
— p58 —
— p48 —
39 —
M
PP
c
b
3
4
➝
2
➝
1
dT18
dT12
5
Figure 1 Phosphorylated DNA polymerase a-primase by Cyclin-Cdk complexes. (a) DNA polymerase a-primase (lane PP) was
incubated with puri®ed Cyclin-Cdk complexes, separated by SDS gel electrophoresis, and stained with Coomassie Brilliant-blue. The
subunits, that were phosphorylated in the presence of Cyclin A-Cdc2 (lane 1), Cyclin A-Cdk2 (lane 2), Cyclin B-Cdc2 (lane 3), Cyclin
E-Cdk2 (lane 4), Cdc2 (lane 5), Cdk2 (lane 6), and control protein (lane 7), were detected by autoradiography. (b) Equal amounts (0.4
units of primase) of DNA polymerase a-primase were pre-incubated with indicated amounts of Cyclin A-Cdc2 (~), Cyclin A-Cdk2
(&), Cyclin B-Cdc2 (*), Cyclin E-Cdk2 (~), Cdk2 (&), or control protein (6), and then primase activities were determined. (c)
DNA polymerase a-primase was treated with Cyclin A-Cdk2 or control protein, and then primase products of pre-treated DNA
polymerase a-primase on poly (dT) ssDNA were analysed by denaturing gel electrophoresis. Lane 1 and 2 Cyclin A-Cdk2-treated
DNA polymerase a-primase (0.2 and 0.4 primase units, respectively), lane 3 and 4 DNA polymerase a-primase treated with control
protein (0.2 and 0.4 primase units, respectively), and 0.4 primase units of untreated DNA polymerase a-primase (lane 5)
to two- to threefold of that of control enzyme (Figure
1b, data not shown). The length of the primase
products was not altered by phosphorylation, since
phosphorylated and untreated DNA polymerase aprimase as well as DNA polymerase a-primase treated
with control protein synthesized equivalent products
(Figure 1c). These data suggest that basic enzymatic
functions of DNA polymerase a-primase were not or
only moderately altered by phosphorylation with Cdks.
Cyclin A-Cdc2 and Cyclin A-Cdk2 inhibit initiation of
SV40 DNA replication in vitro
To study whether the initiation step of replication on
SV40-origin
containing
double-stranded
DNA
(dsDNA) is modulated by phosphorylation, CyclinCdk complex or control protein were added directly to
the initiation reaction. In this assay, Cyclin A-Cdc2 or
Cyclin A-Cdk2 signi®cantly reduced the initiation
Regulation of DNA replication by phosphorylation
C Voitenleitner et al
2
5
6
7
8
– –
– –
units
dT18
dT12
control
Cyclin E-Cdk2
Cyclin B-Cdc2
Cyclin A-Cdk2
control
Cyclin E-Cdk2
Cyclin B-Cdc2
Cyclin A-Cdk2
3 4
+ – +
0.4 0.2 0.4 0.2 0.4 0.2 0.4 0.2 0.4
dT18
➝
1
Arb units
Rel Inc.(%)
+ –
➝
– + –
➝
active Cdk
➝
Cyclin A-Cdc2
1613
dT12
9 10 11 12
41 5.3 29 4.9 33 23 25 27 22 22 23
18 100 15 100 91100124100 95100
Figure 2 Initiation of SV40 DNA replication in the presence of
Cyclin-dependent kinases. The initiation reaction of dsDNA in
the puri®ed system was performed in the presense of
immunoanity puri®ed Cyclin A-Cdc2 (lane 2 and 3), Cyclin
A-Cdk2 (lane 4 and 5), Cyclin B-Cdc2 (lane 6 and 7), Cyclin ECdk2 (lane 8 and 9), or proteins from mock infected cells that
bind to 12CA5 antibodies (control protein, lane 10 and 11). In
parallel, the initiation reactions were performed with either active
(lanes 2, 4, 6 and 8), heat inactivated (10 min at 708C) kinase
complexes (lanes 3, 5, 7 and 9), or with control protein
(untreated, lane 10, and heat treated, lane 11). Reaction products
were analysed by denaturing gel electrophoresis and autoradiography. The initiation products of 0.4 units of untreated DNA
polymerase a-primase are shown in lane 1. Radioactive material
that is detectable in the absence of DNA polymerase a-primase is
shown in lane 12. The length of 5'-end labeled oligo(dT12 ± 18) is
indicated at the right by arrows. The amount of initiation
products was determined by microdensity scanning of the
autoradiograph and displayed at arbitary units (arb units). The
amount of unspeci®c radioactive material (lane 12; 0.36 arb units)
was subtracted. In addition, the amount of initiation products in
the presence of active kinase was normalized to that in the
presence of inactive kinases
activity of DNA polymerase a-primase to about 15%
and 18% of the activity of the inactivated kinase which
served as positive control (Figure 2, lanes 2 to 5). In
the presence of Cyclin B-Cdc2 the initiation reaction
was slightly reduced to about 90% of that in the
presence of heat-treated kinase (Figure 2, lanes 6 and
7), while with the control protein the amounts of
initiation product stayed nearly unchanged (Figure 2,
lanes 10 and 11). In the presence of Cyclin E-Cdk2 the
initiation activity reproducibly increased to about
120% of the level that was determined in the presence
of heat-treated kinase (Figure 2, lanes 8 and 9). The
assay that did not contain kinase or control protein
showed reproducibly higher initiation activity than
those that contained kinase or control protein. Since
this decrease of initiation activity varied with protein
preparations, the initiation activity in the presence of
active kinase was compared to that in the presence of
heat-inactivated kinase.
To study whether phosphorylation of DNA polymerase a-primase a€ects initiation of DNA replication,
we pre-phosphorylated DNA polymerase a-primase
with Cyclin-Cdks, and then both enzyme complexes
were completely separated from each other. Preincubation of DNA polymerase a-primase with
1
2 3
4
5
6
7
8
9 10
M
Arb units 69 0.7 4 18 41 28 58 30 66
Rel Inc.(%)104 2.3 6 60 63 92 88 100 100
Figure 3 Initiation of SV40 DNA replication in vitro by
phosphorylated DNA polymerase a-primase. For comparing the
initiation activity of the DNA polymerase a-primase complexes,
primase activity of each DNA polymerase a-primase was
measured shortly before the initiation assay. The initiation
products of 0.4 units of untreated immunoanity-puri®ed DNA
polymerase a-primase were analysed (lane 1). In parallel, 0.2 and
0.4 primase units of Cyclin A-Cdk2- (lane 2 and 3), Cyclin BCdc2- (lane 4 and 5), or Cyclin E-Cdk2- (lane 6 and 7)
phosphorylated DNA polymerase a-primase, or DNA polymerase a-primase treated with control protein (lane 8 and 9) were
incubated under identical conditions and the initiation products
were analysed. The radioactive material that is detectable in the
absence of DNA polymerase a-primase is shown in lane 10. Lane
M shows 5' end labeled oligo(dT12 ± 18) markers as indicated at the
right by arrows. The amount of initiation products was
determined by microdensity scanning of the autoradiograph and
displayed as arbitary units (arb units). The amount of unspeci®c
radioactive material (lane 10; 0.41 arb units) was subtracted. In
addition, the amount of initation products synthesized in the
presence of 0.2 primase units (lanes 2, 4, 6 and 8) was normalized
to those in lane 8, and the amount of products synthesized by
enzyme equivalent to 0.4 primase units (lanes 1, 3, 5, 7 and 9) was
normalized to those in lane 9
control protein did not in¯uence the initiation of
DNA replication (Figure 3, compare lanes 1 and 9),
since the amount of initiation products that was
synthesized by 0.4 primase units of untreated or
pretreated DNA polymerase a-primase showed about
4% di€erence between both reactions. However, the
amounts of initiation products synthesized by Cyclin
A-Cdk2-phosphorylated DNA polymerase a-primase
decreased to about 2 to 6% of those synthesized by the
control-treated DNA polymerase a-primase (Figure 3,
lanes 2, 3, 8 and 9), and the amount of radioactive
products was only slightly above that of the negative
control in Figure 3 lane 10. In contrast, the Cyclin ECdk2-phosphorylated DNA polymerase a-primase
initiated DNA replication nearly as eciently as the
controls in this experiment, but showed slightly higher
initiation activity in other experiments (Figure 3, lanes
6 to 9; data not shown). In the experiments presented
here, the Cyclin E-Cdk2-treated DNA polymerase aprimase synthesized a high amount of products with
short length, while the lane with control treated DNA
polymerase a-primase contained more long products,
Regulation of DNA replication by phosphorylation
C Voitenleitner et al
1614
which are also initiation products under the assay
conditions (Figure 3, lanes 6 to 9; Stadlbauer et al.,
1996). DNA polymerase a-primase that was prephosphorylated by Cyclin B-Cdc2 showed a moderately reduced initiation activity (reduction to about
two-thirds of that of the control reactions) compared
to that of DNA polymerase a-primase incubated with
control protein (Figure 3, lanes 4, 5, 8 and 9).
Discussion
In this report, we demonstrated that four highly
puri®ed Cyclin-Cdk complexes phosphorylated in vitro
the p180 and p68 subunits of DNA polymerase aprimase, but not the primase subunits (Figure 1a;
Nasheuer et al., 1991). The phosphorylation of DNA
polymerase a-primase barely in¯uenced its basic
enzymatic functions (Figure 1b and 1c; data not
shown). These ®ndings are in agreement with previous
published results that phosphorylation does not or only
modestly change enzymatic properties of DNA
polymerase a-primase (Nasheuer et al., 1991; Podust
et al., 1990; Prussak and Tseng, 1989). These results
suggest that the primase and DNA polymerase activity
that are required for Okazaki fragment synthesis
during lagging strand synthesis are probably not
modulated by Cdk-dependent phosphorylation.
A di€erent picture emerged, when the initiation
activity of DNA polymerase a-primase was examined
on SV40-origin containing dsDNA: Cyclin A-Cdc2
and Cyclin A-Cdk2 inhibited the initiation reaction
(Figure 2, lanes 2 to 5). In contrast, the initiation
activity of DNA polymerase a-primase was only
slightly diminished by Cyclin B-Cdc2, and was even
stimulated by Cyclin E-Cdk2 (Figure 2, lanes 6 to 9).
Inhibition and stimulation of the initiation reaction by
Cdks in vitro were most likely not due to phosphorylation of RP-A, since mutation of Cdk phosphorylation sites RP-A did not change the function of RP-A
in DNA replication (Brush et al., 1994; Henricksen
and Wold, 1994).
To determine whether the DNA polymerase aprimase is regulated by Cdks, the enzyme complex
was preparatively phosphorylated by these kinases.
Although primase of phosphorylated DNA polymerase
a-primase was fully active on ssDNA (Figures 1c, data
not shown), Cyclin A-Cdk2-phosphorylated DNA
polymerase a-primase was no longer active in the
initiation reaction (Figure 3, lanes 2 and 3). These
results suggest that DNA polymerase a-primase might
be controlled by phosphorylation and might catalyse
the initiation reaction on dsDNA and the discontinuous synthesis on ssDNA in di€erent modes. The later
interpretation is supported by studies of species-speci®c
viral DNA replication (BruÈckner et al., 1995; Schneider
et al., 1994; Stadlbauer et al., 1996).
Our ®ndings suggest that SV40 DNA replication is
under phosphorylation control. In vivo, however, SV40
DNA replication occurs in an ampli®cation mode
making an inhibitory control obviously unnecessary.
This apparent discrepancy can eventually be explained
by assuming that papovaviruses have developed
functions to escape phosphorylation control of cellular
proteins, either by inhibiting negatively acting kinases,
or by activating phosphatase(s) that counteract these
kinases. In vitro the viral initiation reaction was under
negative control; viral properties to eliminate initiation
control by phosphorylation might be absent or inactive
in this assay composed of puri®ed proteins only. To
overcome some regulatory functions at the initiation
step, the viruses have evolved an additional strategy:
The virus supplies one essential initiation protein, the
viral T antigen, and therefore becomes partially
independent from the host initiation machinery and
its regulation. Studies to investigate implications of the
presented ®ndings on viral and cellular DNA replication in vivo are currently underway.
Materials and methods
Protein puri®cation
The S. pombe protein suc1 was puri®ed from lysates of
E. coli BL21 containing pRK172-suc1 (Moreno et al.,
1989). The cells were disrupted by sonication, the supernatant of the centrifugated lysates (15 min 20 000 g) was
adjusted to 30% ammonium sulphate, and was applied to
50 ml of phenyl Sepharose (Pharmacia, Freiburg (Germany)). After washing 300 ml of 0.85 M ammonium
sulphate (pH 7.8) the bound proteins were eluted by a
gradient from 0.85 M to 0 M ammonium sulphate. Proteins
in pooled fractions were precipitated with ammonium
sulphate (65%), and then applied to gel ®ltration
(Sephacryl S100, Pharmacia).
For the puri®cation of Cyclin-Cdk complexes 2.56108 to
36108 High Five (ITC Biotechnology GmbH, Heidelberg),
SF9, or SF9X cells were infected with 10 p.f.u./cell of each
recombinant baculovirus and incubated for 44 to 48 h at 278C
(Rosenblatt et al., 1992; Desai et al., 1992). Cells were
homogenized in lysis bu€er (50 mM HEPES-KOH pH 7.5,
100 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM NaF, 5 mM
EGTA, 2 mM EDTA, 1 mM DTT, 0.2% Nonidet-P40, 0.1 mM
Leupeptin, 1% Trasylol1; a generous gift of Bayer Leverkusen
(Germany)) and the complexes were puri®ed by immunoaffinity chromatography with the monoclonal antibody 12CA5
Sepharose 4B or by anity chromatography with suc1Sepharose 4B using 4 mg/ml of HA-peptide or suc1 for
elution. The complexes were dialyzed against 50 mM KPi pH
7.5, 1 mM EDTA, 1 mM 2-mercaptoethanol, 10% glycerol and
stored at 7808C. For control of background activities,
proteins were puri®ed by suc1-anity or immunoanity
chromatography from mock infected cells (control protein).
DNA polymerase a-primase was prepared as described
before (Stadlbauer et al., 1994) with slight modi®cations.
After binding to monoclonal antibody SJK 237-71 Sepharose (Tanaka et al., 1982), the enzyme complex was
extensively washed with 50 mM Tris ± HCl, pH 8.6,
400 mM NaCl, 150 mM KCl, and 1 mM EDTA. For prephosphorylation this resin was equilibrated to histone-kinase
bu€er (20 mM HEPES/KOH pH 7.5, 1 mM DTT, 10 mM
MgCl2, 4 mM EGTA, pH 7.8, 5 mM NaF, 1 mM EDTA, 0.1
mg/ml bovine serum albumine (BSA), 0.1 mM ATP) and
incubated for 30 min at 378C with kinase complexes that
incorporate 50 mmol32P on histone H1 per h. The resin was
washed twice with tenfold column volume of bu€er
containing 50 mM Tris HCl, pH 8.6, 150 mM KCl, 1 mM
EDTA, to remove kinase activity. The enzyme complex was
eluted by pH shift.
Bovine RP-A, Topoisomerase I, and SV40 T antigen were
puri®ed according to Nasheuer et al. (1992) and Moare® et
al., (1993). The DNA polymerase and primase activity were
measured using activated DNA and poly(dT) ssDNA as
previously described (Nasheuer and Grosse, 1987, 1988).
Analysis of primase products was performed as described
Regulation of DNA replication by phosphorylation
C Voitenleitner et al
(Nasheuer and Grosse, 1988). Initiation reactions were
performed according to BruÈckner et al., (1995), and
Stadlbauer et al. (1996), additionally all assays contained
5 mM NaF.
Kinase assays
For kinase assays 1 mg DNA polymerase a-primase was
incubated for 15 min at 378C in histone-kinase bu€er that
included 1 mCi g-[32P]-ATP per assay and kinase complex as
indicated. The proteins were separated by SDS ± PAGE
and the incorporation of phosphate was visualized by
autoradiography. For quantitative results the protein
bands were excised and the radioactivity was measured
by liquid scintillation counting.
Acknowledgements
We thank F Grosse, F MuÈller, and S Dehde for critical
reading the manuscript; K Weiûhart for sharing proteins,
and for useful comments on the manuscript; A Brunahl,
V FoÈrster, and M Hauser for excellent technical assistance;
D Morgan for generous gift of baculoviruses expressing
human Cdc2, Cdk2, Cyclin A, Cyclin B or Cyclin E; J
Schneider-Mergener for providing HA-peptide; for providing baculoviruses expressing human p180, p68, p58 and
p48, we thank T Wang, C Rehfuess, and A BruÈ ckner,
respectively. The ®nancial support of the Deutsche
Forschungsgemeinschaft (Fa 138/5-2, 6-1, Na 190/6-3,
and WI 319/11-2 to E Fanning and H-P Nasheuer), NIH
(1RO1 GM52948-01) to EF and European Community
(CHRX-CT93-0248 DG 12) is gratefully acknowledged.
References
BruÈckner A, Stadlbauer F, Guarino LA, Brunahl A,
Schneider C, Rehfuess C, Prives C, Fanning E and
Nasheuer H-P. (1995). Mol. Cell. Biol., 15, 1716 ± 1724.
Brush GS, Anderson CW and Kelly TJ. (1994). Proc. Nat.
Acad. Sci. USA, 91, 12520 ± 12524.
Challberg M and Kelly TJ. (1989). Annu. Rev. Biochem., 58,
671 ± 717.
Desai D, Gu Y and Morgan DO. (1992). Mol. Biol. Cell., 3,
1021.
Din S-U, Brill SJ, Fairman MP and Stillman B. (1990). Genes
Dev., 4, 968 ± 977.
D'Urso G, Marraccino RL, Marshak DR and Roberts JM.
(1990). Science, 250, 786 ± 791.
Dutta A and Stillman B. (1992). EMBO J., 11, 2189 ± 2199.
Fanning E. (1994). Trends Cell. Biol. 4, 250 ± 255.
Fanning E and Knippers R. (1992). Annu. Rev. Biochem., 61,
55 ± 85.
Ferrari M, Lucchini G, Plevani P and Foiani M. (1996). J.
Biol. Chem., 271, 8661 ± 8666.
Foiani M, Libergi G, Lucchini G and Plevani P. (1995). Mol.
Cell. Biol., 15, 883 ± 891.
Henricksen LA and Wold MS. (1994). J. Biol. Chem., 269,
24203 ± 24208.
Hurwitz J, Dean FB, Kwong AD and Lee S-H. (1990). J.
Biol. Chem., 265, 18043 ± 18046.
Li JJ and Kelly TJ. (1984). Proc. Natl. Acad. Sci. USA, 81,
6973 ± 6977.
McVey D, Brizuela L, Mohr I, Marshak DR and Gluzman
Y. (1989). Nature (London), 341, 503 ± 507.
Moare® I, Small D, Gilbert I, HoÈpfner M, Randall S,
Schneider C, Russo AAR, Ramsperger U, Arthur AK,
Stahl H, Kelly TJ and Fanning E. (1993). J. Virol., 67,
4992 ± 5002.
Moreno S, Hayles J and Nurse P. (1989). Cell, 58, 361 ± 372.
Nasheuer H-P and Grosse F. (1987). Biochemistry, 26,
8458 ± 8466.
Nasheuer H-P and Grosse F. (1988). J. Biol. Chem., 263,
8981 ± 8988.
Nasheuer H-P, Moore A, Wahl AF and Wang TS-F. (1991).
J. Biol. Chem., 266, 7893 ± 7903.
Nasheuer H-P, Winkler DV, Schneider C, Dornreiter I,
Gilbert I and Fanning E. (1992). Chromosoma, 102, S52 ±
S59.
Podust V, Bialek G, Sternbach H and Grosse F. (1990). Eur.
J. Biochem., 193, 189 ± 193.
Prussak CE and Tseng BY. (1989). Biochem. Biophys. Res.
Commun., 159, 1397 ± 1403.
Rosenblatt J, Gu Y and Morgan DO. (1992). Proc. Natl.
Acad. Sci. USA, 89, 2824 ± 2828.
Schneider C, Weiûhart K, Guarino LA, Dornreiter I and
Fanning E. (1994). Mol. Cell. Biol., 14, 3176 ± 3185.
Stadlbauer F, Brueckner A, Rehfuess C, Eckerskorn C,
Lottspeich F, FoÈrster V, Tseng BY and Nasheuer H-P.
(1994). Eur. J. Biochem., 222, 781 ± 793.
Stadlbauer F, Voitenleitner C, BruÈckner A, Fanning E and
Nasheuer H-P. (1996). Mol. Cell. Biol., 16, 94 ± 104.
Stillman B. (1989). Ann. Rev. Cell Biol., 5, 197 ± 245.
Tanaka S, Hu S-Z, Wang TS-F and Korn D. (1982). J. Biol.
Chem. 257, 8386 ± 8390.
Waga S, Bauer G and Stillman B. (1994). J. Biol. Chem., 269,
10923 ± 10934.
Wang TS-F. (1991). Ann. Rev. Biochem., 60, 513 ± 552.
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