Eukaryotic topoisomerase I (Topo I) (
)is capable of
relaxing both negatively and positively supercoiled DNA. The enzyme
catalyzes changes in the superhelical state of duplex DNA by
transiently breaking a single strand, allowing for unwinding of
positively supercoiled DNA or rewinding of negatively supercoiled DNA
(reviewed in (1) ). No metal cation or energy cofactor is
required for Topo I activity, although Mg
and
Ca
, as well as the polycation spermidine, have been
shown to stimulate
activity(2, 3, 4, 5) .
Phosphodiester bond energy is preserved during the nicking-closing
cycle by the formation of a phosphotyrosine bond between the
active-site tyrosine and the 3`-end of the broken strand (6, 7, 8) . This covalent intermediate can be
trapped by denaturing the enzyme during catalysis with either SDS or
alkali(9, 10, 11) . Sequence analyses of a
large number of SDS-induced breakage sites indicated that the cellular
Topo I enzymes will cleave at specific
sequences(9, 12, 13) , but there is only
limited sequence similarity between such sites (9, 12, 13, 14, 15, 16, 17) .
The SDS-induced cleavage at many breakage sites is enhanced by
camptothecin, a plant alkaloid that inhibits the cellular enzymes by
reversibly binding to the covalent Topo I-DNA intermediate in a manner
that slows the the religation step of catalysis (17, 18, 19, 20, 21, 22) .
The human Topo I is composed of 765 residues with a predicted
molecular mass of 91 kDa. Sequence comparisons of cellular eukaryotic
Topo I proteins demonstrate that the human Topo I can be divided into
four domains (Fig. 1). (
)Residues
Met
-Lys
(24 kDa) comprise the
unconserved NH
-terminal domain, which is highly charged
(Asp + Glu = 27%; His + Lys + Arg = 68%)
and contains four putative nuclear localization signals(24) .
Residues Glu
-Ile
(54 kDa) form the
conserved core domain, which is followed by a short positively charged
linker domain of unconserved residues
Asp
-Glu
(5 kDa). Finally, residues
Gln
-Phe
(8 kDa) make up the highly
conserved COOH-terminal domain, which contains the active site tyrosine
at position 723(25, 26) .
Previous reports
have demonstrated that the NH
-terminal domain is sensitive
to proteolyis (3) and that residues 1-230 can be removed
with little if any consequence for Topo I
activity(25, 27) . In contrast, a 5-amino acid
deletion from the COOH terminus abolishes activity. (
)
Figure 1:
Domain
structure of Topo I based on sequence comparisons. Based on amino acid
sequence comparisons of cellular eukaryotic Topo I proteins,
the human enzyme can be divided into four domains. Listed below each domain is the calculated molecular mass for that
domain. Filled areas represent regions that are highly
conserved, while open areas represent the unconserved regions.
Residues Met
-Lys
(24 kDa) comprise the
unconserved amino-terminal domain, which has an unusually high
percentage of negatively and positively charged residues indicated by
the plus and minus signs. Black circles indicate the locations of four potential nuclear localization
signals (residues Lys
-Glu
,
Lys
-Asp
,
Lys
-Asp
, and
Lys
-Glu
)(24) . Residues
Glu
-Ile
(54-kDa) form the conserved
core domain (stippled area). Residues
Asp
-Glu
form an unconserved linker
domain (5 kDa), which is followed by the conserved COOH-terminal
domain, residues Gln
-Phe
(8 kDa),
which contains the active site tyrosine at position
723.
We
have used the baculovirus-infected insect cell system to overproduce
full-length, as well as NH
- and COOH-terminal deletions of
human topoisomerase I. The purified recombinant Topo I is by all
biochemical criteria tested identical to the native enzyme purified
from human cells. Furthermore, we find the activities of the
full-length protein and an amino-terminally deleted enzyme (Topo70,
missing residues 1-174) are identical in every respect. The two
proteins are inhibited by camptothecin and its derivatives, are
stimulated by Mg
with a magnitude that is inversely
proportional to the salt concentration (up to 250 mM KCl), and
are not affected by ATP. By comparing the circular dichroism spectra
and hydrodynamic properties of Topo70 and the full-length protein, we
demonstrate that the amino-terminal domain is largely if not completely
unfolded, while the remainder of the molecule is relatively globular.
In the accompanying paper(40) , we use limited proteolysis to
further examine the domain structure of human Topo I.
EXPERIMENTAL PROCEDURES
Plasmids
All plasmids were constructed by common subcloning techniques
and propagated in either the Sure
(Stratagene) or TOP10F`
(Invitrogen) strains of Escherichia coli. Nucleotides of the
cDNA clone encoding human topoisomerase I are numbered according to
D'Arpa et al.(25) .
pAc-Topo I and pAc-Topo I(Y/F)
The wild type and active
site mutant Y/F human Topo I cDNA sequences from the plasmids pKM16 and
pKM18, respectively(26) , were inserted into the blunt-ended NheI cloning site of pBlueBac (Invitrogen) to generate
pAc-Topo I and pAc-Topo I(Y/F). Restriction analyses were carried out
to confirm proper orientation of the Topo I reading frames with respect
to the polyhedrin gene promoter.
pTopo70-start
The polymerase chain reaction was
used to amplify a segment of the human Topo I cDNA from position 731 to
1112. The 5` end of the amplification product contained an XbaI restriction site followed by an initiating methionine
codon immediately adjacent to the codon for residue Lys
.
The polymerase chain reaction product was digested with XbaI
(which cuts 18 base pairs upstream from the initiating ATG) and NdeI (which cuts at position 901 in the Topo I cDNA) to
generate a 194-base pair fragment that was subcloned into the plasmid
pET11a (Novagen) to generate pTopo70-start. The subcloned sequences
that had been subjected to polymerase chain reaction were sequenced to
confirm proper construction of the initiation signal and to ensure that
no other mutations were present.
pET11a-Topo70
The plasmid pTopo70-start was
linearized with HindIII, which cuts the pET11a vector backbone
at a site just downstream of the Topo I sequence. The HindIII
5` overhangs were filled in with T4 DNA polymerase and all four dNTPs.
This DNA was digested with NdeI, which cuts at position 901 in
the Topo I coding sequences. The resulting fragment was ligated with a
2.7-kilobase pair NdeI
-NotI
(blunt-ended) fragment, which contains the NdeI
-EcoRI
Topo I
sequences followed by multiple cloning site sequences from EcoRI to NotI of the plasmid pSK+ (Stratagene).
The resulting plasmid, called pET11a-Topo70, carries the coding
sequence for a
70-kDa NH
-terminally truncated form of
Topo I called Topo70 that starts at a methionine immediately 5` of
Lys
and ends at the natural Topo I COOH terminus.
pAc-Topo70
The baculovirus transfer vector
pBlueBac (Invitrogen) has a unique NheI cloning site, located
immediately downstream of the polyhedrin gene promoter, into which we
inserted an XbaI-XbaI fragment from pET11a-Topo70,
which carries the entire Topo70 coding sequence. Diagnostic restriction
digests were performed to confirm proper orientation of the Topo70
reading frame with respect to the polyhedrin gene promoter. The
resulting plasmid is named pAc-Topo70.
pAc-Topo70(Y/F)
The NdeI
-AvrII
sequences
from pKM18 (26) were exchanged for the NdeI
-AvrII
sequences of
pAc-Topo70, to generate pAc-Topo70(Y/F). This plasmid is identical to
pAc-Topo70 except that it carries a mutation that converts the active
site tyrosine 723 codon to a phenylalanine codon.
pAc-Topo58
A self-complementary NheI-stop
oligonucleotide (5`-CTAGCCTAGGCCTAGG-3`) was inserted at the unique NheI
restriction site of the plasmid pAc-Topo70
to generate pAc-Topo58. The NheI-stop oligonucleotide
introduces a diagnostic AvrII restriction site in place of the NheI site and positions a stop codon immediately 3` of the
codon for residue Ala
of the Topo I cDNA.
Plasmid Relaxation Assays
Unless stated otherwise, stocks of protein were serially
diluted 2-fold in standard buffer (150 mM KCl, 10 mM Tris-hydrochloride, pH 7.5, 1 mM DTT, 1 mM EDTA,
0.1 mg/ml BSA), and reactions were initiated by the addition of 5
µl of the diluted enzyme to 15 µl of the appropriate buffer
containing 0.5 µg of supercoiled pKSII+ plasmid substrate
(Stratagene). The final reaction conditions are indicated in the table
and figure legends. The reactions were incubated at 37 °C for 10
min and terminated with 5 µl of a stop mix containing 2.5% SDS, 15%
Ficoll, 0.03% bromphenol blue, 0.03% xylene cyanol, and 25 mM EDTA. The products were fractionated by 0.8% agarose gel
electrophoresis and visualzed by ethidium bromide staining. The
inhibitors camptothecin (Sigma), topotecan (NCI, National Institutes of
Health), and 9-amino-camptothecin (NCI) were dissolved in
Me
SO and stored at -20 °C.
Isolation of Recombinant Baculoviruses and Large Scale Sf9
Infection
Recombinant baculoviruses were generated by
co-transfecting Spodoptera frugiperda (Sf9) cells with
linearized wild type Autographica californica multiple
nucleocapsid nuclear polyhedrosis virus DNA (Invitrogen) together with
transfer vector DNAs, and plaque purified according to standard
procedures provided by Invitrogen. To confirm that the recombinant
viruses were expressing the appropriate protein, infected cells were
analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and
immunoblotting. Sf9 cells were maintained in TC100 medium (Life
Technologies, Inc.) supplemented with 10% fetal calf serum, yeastolate
(3.3 g/liter), lactalbumin hydrolysate (3.3 g/liter), 100 units/ml of
penicillin, 100 µg/ml of streptomycin, and 100 units/ml of
nystatin. Cells were cultured in 100 ml and 1-liter spinner flasks
(Bellco) and stirred at a rate of 60 rpm in an atmosphere of 50%
O
, 50% air at 27 °C. The 1-liter flasks were assembled
with microcarrier impellers (Bellco) that were adjusted to break the
air-liquid interface. (
)The maximum volume of medium used in
the spinner flasks was 80 and 500 ml for the 100-ml and 1-liter flasks,
respectively. Typically, cells were seeded at 0.5-0.8
10
cells/ml and cultured until the density reached
3-3.5
10
cells per ml (
3 days). To
infect the cells, they were first pelleted by centifugation at 600
g at room temperature for 5 min and then resuspended
in fresh medium at a density of 1
10
cells/ml. The
appropriate volume of virus stock was added to ensure a multiplicity of
infection of approximately 10 plaque-forming units/cell. After stirring
for 1 h at room temperature, fresh medium was added to the cells such
that the final density was 3
10
cells/ml.
Protein Purification
Purification of Topo I from Baculovirus-infected Insect
Cells
All purification steps except those involving room
temperature high pressure liquid chromatography (Mono Q, Mono S, and
POROS columns) were carried out at 4 °C. At 48 h postinfection,
approximately 3
10
Sf9 cells were harvested by
centrifugation for 5 min at 400
g. The cells were
resuspended in 1 liter of ice-cold phosphate-buffered saline and
centrifuged for 5 min at 400
g. This wash procedure
was repeated twice with 250 ml of phosphate-buffered saline. The washed
cells were resuspended by vigorous shaking in 40 ml of lysis buffer (50
mM KCl, 10 mM Tris-hydrochloride, pH 7.5, 2 mM MgCl
, 1% Triton X-100, 15 mM DTT, 0.15 mg/ml
phenylmethylsulfonyl fluoride, 0.05 mg/ml aprotinin). The nuclei were
washed twice in 80 ml of lysis buffer minus Triton X-100 and
resuspended in 40 ml of resuspension buffer (50 mM KCl, 10
mM Tris-hydrochloride, pH 7.5, 2 mM MgCl
,
25 mM DTT, 0.4 mg/ml phenylmethylsulfonyl fluoride, 0.12 mg/ml
aprotinin). The nuclei were adjusted to 10 mM EDTA and then
lysed by the addition of 50 ml of 2
nuclear extraction buffer
(2 M NaCl, 80 mM Tris-hydrochloride, pH 7.5, 20%
glycerol, 2 mM EDTA). The nuclear extract was stirred for 5
min at
200 rpm. With continued stirring, 50 ml of polyethylene
glycol (PEG) buffer (18% PEG 8000, 1 M NaCl, 10% glycerol) was
added dropwise in order to precipitate the DNA(30) . After
stirring for 40 min, the PEG precipitate was pelleted by centrifugation
at 10,000
g for 10 min. The resulting PEG supernatant
was dialyzed against 4 liters of potassium phosphate buffer (PPB) (250
mM KPO
, pH 7.4, 1 mM DTT, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride). The dialyzed
PEG supernatant was clarified by centrifugation at 10,000
g for 10 min and passed over a 10-ml bed volume of phosphocellulose
(Whatman P11) equilibrated with PPB. The P11 column was washed with 50
ml of PPB and then step-eluted with 30 ml of PPB containing 700 mM KPO
, pH 7.4. The P11 eluate was dialyzed overnight
against 2 liters of PPB and passed over a 6-ml bed volume of
phenyl-Sepharose (PS) (Pharmacia) that was equilibrated with PPB. The
Topo I flowed through the PS column and was collected together with a
10-ml wash. An equal volume of water was then added to the PS
flow-through, which was passed over a Mono Q (5H/R; Pharmacia Biotech
Inc.) column that had been equilibrated with K100 buffer (100 mM KPO
, pH 7.4, 1 mM DTT, and 1 mM EDTA). The Topo I flowed through the Mono Q column and was loaded
onto a Mono S column (5H/R) that was equilibrated with K100. The Mono S
column was eluted with a 25-ml 50-200 mM KPO
, pH 7.4, gradient. Topo I eluted as a single major
peak at
150 mM KPO
. The peak Mono S fractions
were pooled, concentrated with an Amicon ultrafiltration cell, dialyzed
into storage buffer (50% glycerol, 10 mM Tris-hydrochloride,
pH 7.5, 1 mM DTT, 1 mM EDTA), and stored at -20
°C. The high level expression achieved in the baculovirus-infected
insect cell system yielded
10 mg of protein from 3
10
cells.
Purification of Recombinant Topo70 and Topo58
The
purification of recombinant Topo70 was as described above for the
full-length Topo I. The initial steps in the purification of
recombinant Topo58, up to and including elution of the P11 column, were
as described for the full-length enzyme. The P11 eluate was then
dialyzed against K100, filtered through a 0.45-µm syringe filter,
and loaded onto a POROS SP20 (4.6/100) column (PerSeptive Biosystems)
that was equilibrated with K100. The POROS SP20 column was eluted with
a linear 100-500 mM KPO
, pH 7.4, gradient,
and Topo58 was found to elute at 300 mM KPO
. The
peak fractions were pooled, diluted with an equal volume of water, and
loaded onto a Mono S column that was eluted with a 100-300 mM KPO
, pH 7.4, gradient. Approximately 70% of the Topo58
eluted from the Mono S at 200 mM KPO
, while the
remainder eluted at 250 mM KPO
. The 200 mM peak fractions were pooled, diluted with an equal volume of water,
and passed over a Mono Q column that was equilibrated with K100. The
flow-through fractions were concentrated with an Amicon ultrafiltration
cell, dialyzed into storage buffer and stored at -20 °C.
Purification of HeLa Topo I
The starting material
for purification of native human Topo I was 3
10
HeLa S3 cells that were doubling every 20-24 h in
S-modified Eagle's medium (Life Technologies, Inc.) supplemented
with 10% fetal calf serum, 100 units/ml of penicillin, 100 µg/ml of
streptomycin, and 50 units/ml of nystatin. The initial steps in
purification of HeLa Topo I, up to the point of isolating the clarified
dialyzed PEG supernatant, were identical to that described above for
the recombinant enzyme. The dialyzed PEG supernatant was diluted with
an equal volume of water, filtered through a 0.45-µm filter, and
loaded onto a POROS SP20 (4.6/100) column that was equilibrated with
cation exchange buffer (7 mM MES, 7 mM HEPES, 7
mM sodium acetate, pH 7.5) plus 100 mM NaCl. The SP20
column was eluted with a 30-ml linear salt gradient from 100 to 800
mM NaCl. Plasmid relaxation assays were performed to identify
the peak Topo I fractions, which were found to elute at
700 mM NaCl. The Topo I fractions were pooled, dialyzed against 2 liters
of PPB, and passed over a 2-ml bed volume of PS that was equilibrated
with PPB. The PS flow-through was diluted with an equal volume of water
and loaded onto a Mono S column (5H/R) that was equilibrated with K100.
The Mono S column was eluted as described for the recombinant enzyme,
and the peak fractions were dialyzed into storage buffer and stored at
-20 °C.
Purification of f-Topo70 and f-Topo75 Fragments of
Recombinant Topo I
During a 3-week room temperature incubation
of the clarified PEG supernatant from a preparation of
baculovirus-expressed Topo I, all of the full-length enzyme was
proteolytically cleaved into a roughly equal mixture of 70- and 75-kDa
forms of Topo I (designated f-Topo70 and f-Topo75, respectively). At
this time, the PEG supernatant was clarified by centrifugation at
10,000
g for 10 min and dialyzed against 4 liters of
PPB. The dialyzed PEG supernatant was clarified by centrifugation at
10,000
g for 10 min and loaded onto a POROS SP20
(4.6/100) column that was equilibrated with cation exchange buffer plus
400 mM NaCl. The proteins were eluted with a linear 50-ml NaCl
gradient from 400 mM to 1 M. The f-Topo70 and
f-Topo75 co-eluted at 500 mM NaCl. The peak SP20 fractions
were pooled, dialyzed against 2 liters of K100, and loaded onto a Mono
S column (5H/R) that was equilibrated with K100. The column was eluted
with a 20-ml 100-200 mM KPO
, pH7.4,
gradient. The f-Topo75 eluted at 140 mM KPO
, while
the f-Topo70 eluted at 180 mM KPO
. The peak
fractions of each protein were pooled, dialyzed against Mono Q Buffer
(100 mM NaCl, 1 mM DTT, 1 mM EDTA, 20 mM Tris-hydrochloride, pH 7.5), and passed through a Mono Q column
that was equilibrated with Mono Q buffer. Finally, the purified
f-Topo70 and f-Topo75 fractions were dialyzed into storage buffer and
maintained at -20 °C.
Amino-terminal Sequencing
The f-Topo75 and f-Topo70 proteins were fractionated by
SDS-PAGE and then transferred to Immobilon-P (Millipore) membranes in
10 mM CAPS, pH 10. The proteins were visualized by staining
the membranes with Coomassie Blue. The appropriate bands were excised
and sent to DNA Express (Fort Collins, CO) for amino-terminal
sequencing.
Gel Filtration and Glycerol Gradient Sedimentation
Fast protein liquid chromatography (Pharmacia) gel filtration
analyses were performed at 25 °C with a flow rate of 0.75 ml/min
using a Superose 12 (Pharmacia) column that was equilibrated with gel
filtration buffer (200 mM KPO
, pH 7.4, 1 mM DTT, 1 mM EDTA). Purified protein samples of 10-100
µg were diluted into gel filtration buffer and injected in a total
volume of 200 µl. Elution profiles of both Topo I constructs and
molecular weight markers were monitored by UV absorbance at 280 nm and
analyzed by SDS-PAGE. Glycerol gradient sedimentation analyses were
performed by layering 250-µl samples of protein in 200 mM KPO
, pH 7.4, onto a 3.8-ml linear 10-30%
glycerol gradient containing gel filtration buffer. The gradients were
centrifuged at 50,000 rpm in an SW60 rotor (Beckman) for 16 h at 25
°C. Fractions of 300 µl were collected from the bottom of the
gradient tube through a small puncture and analyzed by SDS-PAGE and
silver staining. The experimental gradients each included carbonic
anhydrase as an internal standard. Parallel gradients were used to
determine the sedimentation profiles of the marker proteins.
Circular Dichroism
Proteins were extensively dialyzed into 10 mM KPO
, pH 7.4. Exact molar concentrations of each
protein were calculated from the A
measurements
of fully denatured protein in 6 M guanidine hydrochloride
using molar extinction coefficients that were predicted by the Genetics
Computing Group (GCG) software. CD spectra were obtained at room
temperature, using a Jasco 3000 spectrapolarimeter with a 0.1-cm path
length cell. The molar ellipticity spectrum for each sample was taken
as the average of 8-12 individual scans. To eliminate the
dichroism that is contributed by the sample buffer, the molar
ellipticity spectrum for the dialysis buffer was subtracted from the
molar ellipticity spectrum for each sample. The molar ellipticity
values were normalized for the concentration of amide bonds in each
sample and then converted to 
values(31) .
SDS-Polyacrylamide Gel Electrophoresis and
Autoradiography
SDS-PAGE was performed according to Laemmli(32) .
Proteins were visualized by Coomassie Blue or silver
staining(33) . Autoradiography was performed by exposing dried
gels to Kodak XAR film.
RESULTS
Expression and Purification of Recombinant Forms of
Human Topo I
With the long term goal of investigating the domain
structure of human Topo I, we developed procedures to obtain large
quantities of purified enzyme. This was achieved by the generation of
recombinant baculoviruses that express wild type and active-site mutant
(Y723F) versions of the full-length human Topo I and a 70-kDa protein
(Topo70) which is missing the first 174 NH
-terminal amino
acids (Fig. 2A). The recombinant Topo70 retains at least one
(residues 192-198) of the four potential nuclear localization
signals that reside in the NH
-terminal domain (24) . We also generated a recombinant baculovirus that
expresses a NH
- and COOH-terminally truncated 58-kDa
protein (Topo58), which encompasses the conserved core domain (Fig. 2A). The recombinant proteins have been purified
to apparent homogeneity (Fig. 2B). The wild type
full-length Topo I and Topo70 proteins have the same specific activity
as the native HeLa enzyme (Table 1, line 1). The active site
Y723F mutant protein and Topo58 are at least 5000-fold less active than
the wild type enzyme. The very low level of activity present in the
mutant preparations is due to trace amounts of contaminating insect
cell Topo I.
Figure 2:
Recombinant proteins. Panel A,
baculoviruses were engineered to express the following proteins: wild
type and active-site mutant (Y723F) full-length human Topo I (F.L.
topo I), wild type and Y723F mutant versions of a 70-kDa
NH
-terminally truncated Topo I (Topo70), which initiates
translation with an engineered methionine immediately upstream of
Lys
, and an NH
- and COOH-terminally truncated
58-kDa form of Topo I (Topo58), which has the same initiating
methionine as Topo70 but is terminated after residue Ala
.
The predicted molecular mass (kDa) for each protein is indicated at the right. Panel B, purified proteins (5 µg each)
were fractionated by 9-17% SDS-PAGE and visualized by Coomassie
Blue staining. Lane 1 contained molecular mass markers
(Bio-Rad) myosin (200 kDa),
-galactosidase (116 kDa), phosphoylase
b (97 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (31
kDa), lysozyme (14.4 kDa), and aprotinin (6.3 kDa). Lane 2,
HeLa Topo I. Lane 3, recombinant Y723F full-length Topo I. Lane 4, Y723F proteolytic fragment of 75 kDa (f-Topo75). Lane 5, Y723F proteolytic fragment of 70 kDa (f-Topo70). Lane 6, Y723F recombinant Topo70. Lane 7, recombinant
Topo58.
The recombinant full-length Topo I and HeLa cell Topo I
co-migrate in SDS-PAGE analyses (Fig. 2B, compare lanes 2 and 3). Although the full-length proteins are
predicted to be 91-kDa(25) , they migrate anomalously in SDS
gels with an apparent molecular mass of
100 kDa. In contrast, the
recombinant Topo70 migrates appropriately with an apparent molecular
mass of
70 kDa (Fig. 2B, lane 6). Since
it has previously been observed that negatively charged residues retard
the migration of proteins in SDS gels(34) , it is likely that
the 48 negatively charged residues in the first 174
NH
-terminal amino acids are responsible for the slow
migration of the full-length protein.
While designing the
purification scheme, we noticed that long term storage of nuclear
extracts leads to proteolysis of the full-length enzyme into a
fragments of 75 and 70 kDa, designated f-Topo75 and f-Topo70,
respectively (Fig. 2B, lanes 4 and 5). Both of these fragments were purified and found to display
activity equal to that of the full-length protein (Table 1, line
1), indicating that they retain the active site tyrosine, which is very
close to the COOH terminus. This observation, together with the fact
that amino acids downstream of the active site are essential for
activity(27) ,
indicated that f-Topo75 and f-Topo70
represent NH
-terminally deleted forms of Topo I. Amino
terminal sequencing confirmed that f-Topo75 was missing the first 137
residues while f-Topo70 was missing the first 174 residues.
The Effect of Divalent Metal Cations and Polycations on
Topo I Activity
Although not required for Topo I activity,
divalent metal cations are known to stimulate the activity as much as
25-fold (2, 3) . Therefore to further characterize the
recombinant full-length Topo I and Topo70, we examined the effect of a
variety of divalent cations on the activity of the two proteins. In the
standard relaxation assay buffer (which contains 150 mM KCl),
Mg
, Mn
, Ba
, and
Ca
were all found to stimulate Topo I activity
approximately 16-fold (Table 1, lines 2, 10, 19, 20, and 21). In
contrast, Cd
, Zn
,
Co
, and Cu
completely inhibit Topo
I (Table 1, lines 22-25), while Ni
was found to
inhibit activity 16-fold at 5 mM (Table 1, line 26). To further investigate the stimulatory effect of Mg
we thought it would be informative to determine the level of
Mg
stimulation obtained over a range of salt
concentrations (Fig. 3). Initially, we varied the salt
concentration in the absence of Mg
and found that the
optimal KCl concentration was 200-250 mM, with very
little activity detected at KCl concentrations less than 10 mM or greater than 400 mM (Fig. 3A, and data
not shown). We then assayed the effect of 10 mM Mg
over a range of KCl concentrations from 25 to 350 mM.
This revealed that Mg
had its largest stimulatory
effect (50-fold) at low salt concentrations (25 mM KCl). As
the KCl concentration was increased, the stimulatory effect of
Mg
steadily dropped, and at
250 mM Mg
was slightly inhibitory. With higher salt
concentrations, 300 and 350 mM, the addition of Mg
was 3- and 7-fold inhibitory, respectively. The full-length and
NH
-terminally truncated Topo70 enzymes behaved identically
in their responsiveness to salt and Mg
(data not
shown). For both forms of Topo I, there was an inverse relationship
between the fold-effect of Mg
on activity and the KCl
concentration. This is best depicted graphically as a logarithmic plot
of the ratio of enzyme activity with and without Mg
versus the KCl concentration (Fig. 3B).
Figure 3:
The
effect of Mg
and KCl on Topo I activity. Relaxation
assays were initiated by mixing 10 ng of full-length Topo I (in 4
µl of dilution buffer 150 mM KCl, 10 mM Tris-hydrochloride, pH 7.5, 1 mM DTT, 1 mM EDTA,
0.1 mg/ml BSA) with 130 µl of assay buffer (variable KCl
concentration, 10 mM Tris-hydrochloride, pH 7.5, 1 mM DTT, 1 mM EDTA, 0.1 mg/ml BSA, 0.025 µg/µl
plasmid DNA, with or without 10 mM MgCl
) that had
been prewarmed to 37 °C. Reactions were incubated at 37 °C, and
at various time points 20-µl aliquots were stopped with SDS. The
products were fractionated by 0.8% agarose gel electrophoresis and
visualzed by ethidium bromide staining. Complete relaxation was said to
have been achieved when supercoiled plasmid could no longer be detected
visually. Panel A is a graphical representation of the log of
the reciprocal of the time (min) required to fully relax the plasmid
DNA versus the concentration of KCl. Panel B is a
graphical representation of the log of the ratio (activity in the
absence of Mg
/activity in the presence of
Mg
) versus the concentration of
KCl.
Like divalent metal cations, polycations such as spermine and
spermidine have also been shown to stimulate Topo I
activity(5, 35) . However, previous reports did not
describe the effect of a combination of Mg
and a
polycation, which might shed light on the stimulatory mechanism of each
alone. For example, if the Mg
and spermidine effects
were additive, then this might suggest separate mechanisms for
activation. Thus, we examined the ability of spermidine and spermine to
stimulate Topo I activity in the presence and absence of 10 mM MgCl
(Table 1). In the absence of
Mg
, spermine (1 mM) and spermidine (5
mM) were found to stimulate Topo I activity 8-fold at 150
mM KCl. However, the combination of 10 mM Mg
and 1 mM spermidine stimulated the
activity 64-fold. This suggests that the effects of polycations and
Mg
are at least partially additive and that the two
substances may stimulate Topo I activity by different mechanisms.
Effect of ATP on Topo I Activity
Topo I does not
require ATP or any other energy source for activity. However, it has
been reported that physiological concentrations of ATP (
2
mM) can inhibit human Topo I(36) . In another report,
ATP was shown to inhibit human Topo I only in the presence of 1 mM KPO
(37) . These conflicting reports prompted
us to examine the effect of ATP and KPO
on the activity of
HeLa Topo I in the presence or absence of 10 mM Mg
(Table 1). In the absence of Mg
, 4 mM ATP had no detectable effect on Topo I activity whether 3 mM KPO
was included or not (Table 1, lines 6 and
7). In the presence of 10 mM Mg
, 4 mM ATP was found to inhibit the activity 2-fold regardless of the
presence or absence of 3 mM KPO
(Table 1,
lines 8 and 9). Since ATP was only inhibitory in the presence of
Mg
, it seemed possible that the inhibitory effect of
ATP could be the consequence of its ability to bind
Mg
, which would effectively lower the concentration
of free Mg
. To test this possibility, we assayed the
activity in the presence of 6 mM Mg
, which
is the expected concentration of free Mg
in a mixture
of 10 mM MgCl and 4 mM ATP. Activity in the presence
of 6 mM Mg
was reduced 2-fold relative to
activity in the presence of 10 mM Mg
(Table 1, compare lines 2 and 10). Similar results were
obtained for both recombinant full-length Topo I and Topo70. Thus, we
conclude that the inhibitory effect of ATP is not the result of binding
to the enzyme but rather is the consequence of the ability of ATP to
reduce the free Mg
concentration.
The Effect of Camptothecin and Its Derivatives on Topo I
Activity
To further characterize the activity of the recombinant
full-length Topo I and Topo70, we examined the inhibitory effects of
camptothecin and its derivatives, topotecan and 9amino-camptothecin.
When camptothecin was included in the reactions at 50 µM,
activity was inhibited 8-16-fold in the presence or absence of 10
mM Mg
, 1 mM spermine, or 5 mM spermidine (Table 1, lines 11-14). Topotecan and
9-amino-camptothecin were also found to inhibit relaxation
8-16-fold in the presence or absence of Mg
(Table 1, lines 15-18).
Hydrodynamic Properties of Full-length Topo I and
Topo70
The sensitivity of the NH
-terminal one-fourth
of Topo I to proteolysis suggested that it might be in an extended
conformation, while the remaining three-quarters of the protein, which
is more resistant to proteolysis, might be more globular. To test this
notion, we subjected full-length Topo I, Topo70, and Topo58 to gel
filtration and glycerol gradient sedimentation analyses (Fig. 4). As previously reported(38) , human Topo I
chromatographed through gel filtration with an apparent molecular mass
of
300 kDa (Fig. 4A). In contrast, the same
protein sedimented in a glycerol gradient with an apparent molecular
mass of
66 kDa (Fig. 4B). The discrepancy in the
molecular mass estimates by the two methods suggests that Topo I has an
extended shape and thus a larger frictional coefficient than would be
expected for a globular protein of 91 kDa. In contrast, the
NH
-terminally truncated Topo70 was found to have an
apparent molecular mass of
66 kDa by sedimentation and
96 kDa
by gel filtration, indicating that the NH
-terminal region
is largely responsible for the asymmetric shape of the protein.
Figure 4:
Gel
filtration and glycerol gradient sedimentation. Panel A,
proteins were chromatographed over a Superose 12 column, and elution
profiles were monitored by UV absorbance at 280 nm. Molecular mass
standards (Sigma) were apoferritin (443 kDa),
-amylase (200 kDa),
bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anyhdrase
(29 kDa), and lysozyme (14 kDa). Experimental samples were full-length
recombinant Topo I (F.L. topo I), recombinant Topo70, and
recombinant Topo58. The results are presented graphically as log
(molecular mass) versus the ratio of observed elution volume (Ve) to excluded volume (Vo). The calculated apparent
molecular masses for full-length Topo I, Topo70, and Topo58 are
300,
96, and
83 kDa, respectively. Panel B,
proteins were fractionated by sedimentation through 10-30%
glycerol gradients. Fractions were collected and analyzed by SDS-PAGE
and silver staining. Molecular mass standards (Sigma) were
-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum
albumin (66 kDa), ovalbumin (45 kDa), and carbonic anyhdrase (29 kDa).
The results are presented graphically as molecular mass versus fraction number. The calculated apparent molecular masses for
full-length Topo I, Topo70, and Topo58 are
66,
66, and
60 kDa, respectively.
Since full-length Topo I and Topo70 both co-sediment with BSA in a
glycerol gradient, we can assume that the sedimentation coefficients
for the two proteins are approximately equal to that of BSA
(s
= 4.55)(39) . Thus, the
frictional coefficient (f) for each enzyme can be estimated
from the formula f =m(1 - v
)/s, (where m is molecular mass, v is the partial specific volume,
is the density of the
solvent, and s is the sedimentation coefficient(39) .
The partial specific volumes for full-length Topo I and Topo70 are both
0.74, as predicted from the amino acid content of each
protein(39) . Thus, taking
as equal to 1.0 g/cm
(the density of water at 20 °C), the frictional coefficients
for the full-length protein and Topo70 are calculated to be 8.7
10
and 6.6
10
(g
s
), respectively. Given these
frictional coefficients, the axial ratio (39) of the
full-length protein is approximately 10, as compared with a value of 5
for Topo70 (BSA is 6), confirming the elongated shape for full-length
Topo I.
Topo58 and Topo70 eluted from a gel filtration column with
similar apparent molecular masses of
83 and
96 kDa,
respectively. However they sedimented with apparent molecular masses of
59 and
66 kDa. The
30-kDa disparity between molecular
mass estimates by the two methods indicates that the shared 58 kDa
domain of the two proteins either contains a small highly extended
region or is by itself somewhat elongated. Evidence from limited
proteolysis studies (see accompanying paper(40) ) indicates
that the former possibility is more likely to be correct. For example
limited trypsin digestion of full-length Topo I generates a
proteolytically resistant 55-kDa fragment that starts at residue
Lys
and ends somewhere very close to residue Lys
(Fig. 1A in (40) ). The sensitivity to
proteolysis strongly suggests that the NH
-terminal segment
of the Topo58 protein is likely extended (residues
Lys
-Lys
), providing the hydrodynamic
feature that leads to the
30-kDa discrepancy.
CD studies of Full-length Topo I and Topo70
The
gel filtration and sedimentation analyses indicated that human Topo I
has a highly extended NH
terminus, which minimally includes
the first 174 residues but probably extends up to residue 197. To
further investigate this structural feature, we obtained the far UV CD
spectra for both the wild type and active-site mutant (Y723F) versions
of full-length Topo I and Topo70 (Fig. 5). Within experimental
error, the CD spectra for the wild type and mutant versions of the two
proteins are identical (Fig. 5, A and B).
Hence, as expected the active-site mutation Y723F does not appreciably
alter the structure of the enzyme as measured in this way. In contrast,
the averaged CD spectra of Topo70 and full-length Topo I are different (Fig. 5C). To minimize experimental error in this
comparison, we averaged the mutant and wild type spectra obtained for
two different preparations of each protein. Topo70 has a significantly
stronger polarization in the 190-nm range than the full-length protein.
Since polarization at 190 nm is a very reliable predictor of
-helical content in proteins(31, 41) , it is
immediately apparent that the Topo70 protein has a greater percentage
of
-helical content than the full-length protein. To confirm this
notion, we compared the percentage secondary structures of the two
proteins as predicted by the program VARSLC1(41) . The
predicted molecular mass of
-helix (
28 kDa) and parallel
-sheet (
3 kDa) are very similar for the two proteins.
However, the full-length protein is predicted to contain an extra 10
kDa of unfolded regions, 8 kDa of turns, and 4 kDa of antiparallel
-sheet. Hence, while the NH
terminus appears to be
largely unfolded, it also appears to have a substantial quantity of
turns and antiparallel
-sheet. The turns and antiparallel
-sheet could either be folded into a stable contiguous domain or
instead could be a reflection of transiently formed secondary structure
in an otherwise random coil (41) . To assess which of the
explanations is more likely, we analyzed the first 174 residues Topo I
with the PEPTIDESTRUCTURE function of the GCG software package (Fig. 5D). This program predicts only very short
regions of secondary structure for the NH
-terminal domain.
Taking all of the information into account, gel filtration,
sedimentation, and CD analyses, we conclude that the amino-terminal 174
resides of Topo I are largely unfolded and are comprised of very little
if any extended secondary structure.
Figure 5:
Circular dichroism. CD spectra are
presented graphically as 
values versus wavelength
(nm). Panel A shows representative spectra for wild type (solid line) and active site mutant (Y723F) (dashed
line) versions of full-length Topo I. Panel B shows
representative spectra for wild type (dashed line) and Y723F (solid line) versions of Topo70. Panel C depicts the
average spectra for full-length Topo I (dashed line) and
Topo70 (solid line), obtained by combining and then averaging
mutant and wild type spectra (a total of seven spectra) for different
preparations of each protein. Panel D is a graphical
representation of the Chou-Fasman peptide structure prediction of
residues 1-174 made by the GCG software. Sine waves,
-helix; sharp sawtooth,
-sheet; 180° change
in direction, turns; dull sawtooth, random
coil.
DISCUSSION
The Effects of Metal Cations and Polycations
The
recombinant human Topo I produced in the baculovirus-infected insect
cell system displays the same apparent molecular weight and specific
activity as the native enzyme purified from HeLa cells. Consistent with
earlier findings(3, 25, 27) , the full-length
Topo I and the NH
-terminally truncated Topo70 display
identical activities. Both enzymes are inhibited by camptothecin,
topotecan, and 9-amino-camptothecin but not by ATP. The activities of
both are stimulated by Mg
, Ba
,
Ca
, and Mn
, but are strongly
inhibited by Ni
, Zn
,
Cu
, Cd
, and Co
.
The stimulatory effect of Mg
was found to increase
with decreasing salt concentration. Under low salt conditions of (25
mM), the 50-fold stimulatory effect of Mg
resulted in a final level of activity that was still 10-fold less
than that achieved with the most favorable condition, a combination of
10 mM Mg
and 200 mM KCl. This
suggests that Mg
may stimulate the activity by two
mechanisms, one similar to that achieved by monovalent cations alone
and another that further stimulates activity in the presence of
monovalent cations up to 200 mM KCl. At higher KCl
concentrations (
250 mM) Mg
was found to
be slightly inhibitory, as has been observed for the rat liver and
vaccinia Topo I enzymes(4, 35) .There are several
potential mechanisms whereby a divalent cation such as Mg
could effect a large stimulation of Topo I activity. For example
Mg
is known to effectively shield the negative charge
of the phosphate backbone of duplex DNA, which in addition to allowing
the two strands to wind tighter (42) also reduces the effective
diameter of the double helix(43, 44) , making it more
favorable for two duplexes to lie on top of each other to form a node (45) . Since it has been shown that Topo I has a preference for
binding to nodes(46, 47) , it could be envisioned that
the presence of Mg
-facilitated nodes recruits Topo I
to supercoiled DNA, thereby effectively increasing activity.
Alternatively, Topo I may simply prefer to relax DNA with a
Mg
-shielded phosphate backbone. Another possibility
is that Mg
binds to the enzyme effecting some
allosteric activation. However, the fact that Mg
does
not influence the patterns of limited proteolysis of either the free or
DNA-bound protein (see accompanying paper(40) ), suggests that
Mg
does not have any major effect on enzyme
structure. For the vaccinia Topo I, Mg
has been shown
to stimulate activity by accelerating the release of DNA substrate
following topoisomerization, which is the rate-limiting step in steady
state catalysis under low salt conditions(48) . Since the viral
enzyme has no detectable divalent metal cation binding
site(48) , this effect is presumably mediated by metal cation
binding to DNA. Given the known effects of monovalent cations on
processivity (4) and the similarity between the stimulatory
effects of monovalent cations and Mg
, it seems likely
that the cellular enzymes are stimulated by Mg
in a
manner similar to the viral enzyme. The possibility of direct
participation of a divalent metal cation in phosphodiester bond
cleavage would seem to be excluded by the fact that metal cations are
not required for Topo I activity.
The stimulatory effects of
Mg
and the polycations (spermine and spermidine) were
found to be at least partially additive, suggesting that the two
substances may influence Topo I activity by separate mechanisms.
Spermine and spermidine can effectively shield the phosphate backbone
of duplex DNA by binding in the minor groove(49) . This
information and the fact that certain minor goove binding compounds are
known to be inhibitors of Topo I (50) can be taken to suggest
that the configuration of the minor groove can influence Topo I
activity. This hypothesis is further supported by the observed weak
consensus sequence for Topo I cleavage (5`-(A/T)(G/C)(A/T)T-3`), which
is suggestive of protein contacts within the minor
groove(9, 16) . Alternatively, spermine and/or
spermidine may have an allosteric effect on Topo I. Further experiments
are needed to better define the effects of both divalent and polyvalent
cations on Topo I activity.
The NH
-terminal Domain
The role of the
unconserved NH
-terminal domain remains elusive. All
cellular eukaryotic topoisomerase I enzymes have this highly charged
domain, which in every case examined is dispensable for in vitro activity(3, 24, 27, 30, 51) .
However, since this domain contains nuclear localization signals, it is
nevertheless required for the in vivo function of Topo
I(24) . Of the four putative nuclear localization signals
residing in NH
-terminal domain of the human enzyme,
residues Lys
-Asp
have been suggested
to be the most important for nuclear localization based on sequence
comparisons with other cellular eukaryotic Topo I enzymes(24) .
However, we find that Topo70, which starts at a methionine immediately
5` to residue Lys
, is transported to the nucleus of
insect cells. Hence, the single remaining intact nuclear localization
signal (Lys
-Glu
) must be sufficient
for nuclear transport, at least in insect cells.Aside from its role
in nuclear localization, what is the function of the NH
terminus? In the human enzyme, we find that residues 1-174
of the NH
-terminal domain are largely if not completely
unfolded and that the activity of the Topo70 enzyme (which is missing
these residues) is indistinguishable from that of the full-length
enzyme by every criterion tested. The highly extended nature of the
NH
-terminal domain was observed using analytical techniques
that involved conditions of both low salt (circular dichroism, 10
mM) and high salt (sedimentation and gel filtration, 200
mM). Hence, the extended nature of the NH
terminus
persists under variable salt concentrations and is not an artifact of
any single analytical technique. With its high density of both
negatively and positively charged residues (67% charged residues), the
NH
terminus is almost zwitterionic in nature. Accordingly,
we have observed that the full-length enzyme can be concentrated to
>50 mg/ml with no signs of precipitation. In contrast, Topo70 can
only be concentrated to
5 mg/ml before it begins to precipitate
(data not shown). Hence, the NH
-terminal domain acts as a
solubilizing element in vitro. A conservative estimate for the
quantity of Topo I in HeLa cells can be made based on our recovery of
250 µg of purified Topo I from 10
HeLa cells. Since
greater than 95% of the cell protein is associated with the nuclear
compartment (data not shown), which has an average free water volume of
approximately 3000 µM
for HeLa S3
cells(52) , we estimate that the minimal nuclear concentration
of Topo I is
100 µg/ml. This concentration is 50-fold below
the solubility limit of Topo70. However, Topo I is known to be highly
concentrated in regions of chromatin that are undergoing high levels of
transcription such as activated heat-shock loci and the
nucleolus(28, 29, 53, 54) .
Furthermore, UV cross-linking and SDS-induced trapping of Topo I-DNA
complexes has revealed that Topo I is enriched at least 20-fold on rDNA
relative to total DNA (29) and is enriched 20-fold at induced
heat shock loci relative to the uninduced loci(23) . Thus, the
possibility exists that local concentrations of Topo I could approach 5
mg/ml, which might necessitate a solubility factor such as the
zwitterionic NH
terminus.
The Shape of Topo I
In addition to revealing the
extended nature of the NH
-terminal domain, the comparison
of the hydrodynamic properties of full-length Topo I to those of Topo70
and Topo58 revealed that the core, linker, and COOH-terminal domains
fold into a globular structure. Hence, the catalytically active domain
of Topo I is largely globular, while the NH
-terminal domain
is largely unfolded, consistent with it being dispensable for activity.
Since the Topo58 core domain was capable of folding into a well behaved
globular molecule, it probably represents a subdomain of Topo I that is
capable of folding independent of the amino-terminal, linker, or core
domains. Finally, the availability of milligram quantities of purified
full-length Topo I, Topo70, and Topo58 greatly facilitated further
studies of the structure-function relationships of Topo I. In the
accompanying paper, we describe the use of limited proteolysis to
further characterize the domain structure of the protein(40) .