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(Received for publication, October
12, 1995; and in revised form, January 21, 1996) From the
Using limited proteolysis, we show that the domain boundaries of
human topoisomerase I closely parallel those predicted from sequence
comparisons with other cellular Topo I enzymes. The enzyme is comprised
of (i) an NH
Eukaryotic topoisomerase I (Topo I) ( Sequence comparisons of the
cellular eukaryotic Topo I enzymes ( Weak
amino acid sequence homology exists between the cellular Topo I enzymes
and the the vaccinia viral Topo I (similarity = 43%, identity
= 20%). Two segments of the human enzyme show similarity to the
vaccinia Topo I. These are residues Pro To further examine the domain structure of human Topo I, we
subjected the recombinant enzyme to limited proteolysis with trypsin
and subtilsin. The digestion patterns show that the conserved core and
COOH-terminal domains are globular, tightly folded segments of the
protein, while the NH
Figure 1:
Limited
subtilisin and trypsin digestion of Topo I. A, full-length
Topo I (F.L. topo I) was digested with increasing
concentrations of subtilisin (lanes 2-5) or trypsin (lanes 7-10). The 34-µl reactions contained 10
µg of Topo I plus the following quantities of protease: 0.2 µg (lanes 2 and 7), 0.1 µg (lanes 3 and 8), 0.05
µg (lanes 4 and 9), 0.025 µg (lanes 5 and
10), or no protease (lanes 6 and 11). Aliquots of the
digestion products were fractionated by 5-20% SDS-PAGE and
visualized by Coomassie Blue staining. Lanes 1 and 12 contained a mixture of 5 µg of f-Topo75 and f-Topo70 (see (5) ). Lane 13 contained 10 µg of Topo I that was
not incubated under digestion conditions. Lane 14 contained
the molecular mass markers (Sigma) bovine serum albumin (66 kDa),
ovalbumin (45 kDa), trypsinogen (24 kDa),
The purified oligonucleotides were radiolabeled by
incubating 5 µg of oligonucleotide with 100 µCi of
[
To learn more about the amino
acid sequence content of the Topo I fragments, we subjected s-Topo60
and t-Topo55 to NH To determine the
level of Topo I activity that remains following digestion with
subtilisin, we terminated proteolysis by the addition of PMSF and
performed plasmid relaxation assays with the digested proteins (Fig. 1B). Surprisingly, even when most of the
full-length protein had been converted into the s-Topo60 fragment (sample 2), there was only a
Figure 2:
Subtilisin and trypsin digestion of
Figure 6:
Domain structure of Topo I. As revealed by
the biochemical analyses presented in this and the accompanying
manuscript(5) , the domain structure of human Topo I closely
parallels that which is predicted from sequence comparisons. The
NH
Figure 3:
The effect of plasmid DNA and
Mg
Figure 4:
The effect of duplex oligonucleotide DNA
on subtilisin digestion of Full-length Topo I (F.L. topo I)
and Topo70. A, active site mutant Y723F full-length Topo I was
digested with 2-fold increasing concentrations of subtilisin (starting
at 1 ng/µl) in SDB plus 10 mM MgCl
In the presence or
absence of either DNA or Mg
In general, the digestion patterns of the It should be noted that with high concentrations of
subtilisin, the s-Topo60 core fragment is further digested into at
least one fragment of
Figure 5:
Subtilisin digestion of suicided Topo70 in
the presence or absence of plasmid DNA. Topo70 was incubated with a
In principle, the effects of DNA
binding on proteolysis of Topo I can be explained by either of two
general models. The first model supposes that direct DNA binding to a
given region blocks access of the protease, resulting in resistance to
cleavage. The second model states that DNA binding to one region of the
protein causes a conformational shift, rendering another region more or
less sensitive to proteolysis. The relatively large number of
positively charged residues in the linker domain (15 out of 45,
residues Asp The
observation that the linker domain of permanently trapped covalent
complexes is sensitive to proteolysis both in the presence and absence
of added duplex DNA is not necessarily inconsistent with the direct
binding model if one assumes that the short segment of single-stranded
DNA is insufficient to protect the linker from proteolysis. In this
case, one would have to further assume that the suicided oligo would
sterically prevent the covalent complex from binding to additional
duplex DNA molecules. In an alternative model, it could be envisioned
that upon cleavage of DNA the COOH-terminal and/or core domains undergo
a conformational shift that renders the linker susceptible to
proteolysis in the absence and presence of added DNA. Though the
exact nature of DNA-induced changes that result in differential
protease sensitivity remains unclear, our results indicate that the
linker domain of Topo I can exist in one of two different states. The
linker domain of the free enzyme or of the suicided covalent complex is
in an ``open'' state and is sensitive to proteolysis.
However, when Topo I is noncovalently bound to duplex DNA, the linker
is in a ``closed'' state and is less sensitive to
proteolysis. These results suggest the intriguing possibility that the
linker domain may switch from the ``closed'' to the
``open'' state upon formation of the covalent enzyme-DNA
complex. We are currently investigating this possibility by examining
the proteolytic sensitivity of the linker domain when the enzyme is
noncovalently and covalently complexed to a suicide substrate comprised
of duplex DNA flanking both sides of the cleavage site. The results
from this experiment will also help to resolve which of the two above
models better explains the proteolytic sensitivity of the linker domain
in the suicided covalent complex.
The structural differences
between the human and vaccinia enzymes can be further contrasted when
considering the effects of DNA on limited proteolysis. Covalent and
noncovalent binding of vaccinia Topo I to duplex oligonucleotide
results in 10-fold inhibition of proteolysis at both linker
domains(4) . In the case of the human enzyme, noncovalent
binding to duplex DNA results in protection of the linker domain but
has a negligible effect on proteolysis of the NH
Volume 271,
Number 13,
Issue of March 29, 1996 pp. 7602-7608
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-terminal domain (24 kDa), which is known
to be dispensable for activity, (ii) the core domain (
54 kDa),
(iii) a linker region (
3 kDa), and (iv) the COOH-terminal domain
(
10 kDa), which contains the active site tyrosine. The highly
conserved core and COOH-terminal domains are resistant to proteolysis,
while the unconserved NH
-terminal and linker domains are
sensitive. Noncovalent binding of Topo I to plasmid DNA or to short
duplex oligonucleotides decreases the sensitivity of the linker to
proteolysis by approximately a factor of 10 but has no effect on
proteolysis of the NH-terminal domain. When the enzyme is
covalently complexed to an 18 base pair single-stranded
oligonucleotide, the linker region is sensitive to proteolysis whether
or not duplex DNA is present. The net positive charge of the linker
domain suggests that at a certain point in catalysis the linker may
bind directly to DNA. Further, we show that limited subtilisin cleavage
can generate a mixture of 60-kDa core and 10-kDa COOH-terminal
fragments, which retain a level of topoisomerase activity that is
nearly equal to undigested control samples, presumably because the two
fragments remain associated after proteolytic cleavage. Thus, despite
its potential role in DNA binding, the linker domain (in addition to
the NH
-terminal domain) appears to be dispensable for
topoisomerase activity. Finally, the limited proteolysis pattern of the
human enzyme differs substantially from the limited proteolysis pattern
of the vaccinia viral Topo I, indicating that the two enzymes belong to
separate eukaryotic topoisomerase I subfamilies.
)relaxes both
negatively and positively supercoiled DNA by catalyzing the transient
breakage of a phosphodiester bond in a single DNA strand (reviewed in (1) ). No metal cation or energy cofactor is required for Topo
I activity. Cleavage of a phosphodiester bond in DNA involves a
transesterification reaction in which the nucleophilic O-4 oxygen of
the active site tyrosine (amino acid 723 in human Topo
I(2, 3) ) attacks the phosphodiester linkage. This
results in the formation of a phosphotyrosine bond between the enzyme
and the 3` end of the broken strand. Reversal of the
transesterification reaction restores the phosphodiester bond and
liberates the enzyme. Two models, free rotation and enzyme-bridging,
have been proposed to explain the mechanism by which Topo I promotes
topoisomerization of the DNA (reviewed in (1) ). The free
rotation model proposes that the 5` end of the broken strand is
released from the active site and is allowed to freely rotate about the
unbroken strand. The enzyme-bridging model proposes that the unbroken
strand is passed through an enzyme-bridged break, formed by covalent
attachment to the 3` end of the broken strand and noncovalent binding
to the 5` end of the broken strand.)reveals the human
enzyme (765 residues, 91 kDa) can be divided into four domains (see
preceding paper (5) ): the highly charged (Asp + Glu
= 27%; His + Lys + Arg = 68%) unconserved
NH-terminal domain (residues
Met-Lys, 24 kDa), which contains four
putative nuclear localization signals(6) ; the conserved core
domain (Glu
-Ile
, 54 kDa); a short
unconserved positively charged linker domain
(Asp
-Glu
, 5 kDa); and the highly
conserved COOH-terminal domain
(Gln
-Phe
, 8 kDa) which contains the
active-site tyrosine at position 723 (Refs. 2, 3, and 7).
In the accompanying paper (5) we demonstrate that the
NH-terminal domain is mostly if not completely
unstructured, an observation that is consistent with the fact that this
domain is sensitive to proteolysis and is dispensable for activity (5, 6, 7, 8, 9) .-His
of the core domain and residues
Gly
-Phe
of the COOH-terminal domain,
which includes the active site tyrosine.
Like the cellular
enzymes, vaccinia Topo I relaxes negatively and positively supercoiled
DNA, does not require an energy cofactor or divalent cation, and is
stimulated by Mg(10) . Despite these
similarities, there are major differences between the two enzymes.
First, the vaccinia enzyme is not inhibited by
camptothecin(10) , a plant alkaloid that inhibits the cellular
enzymes by slowing the religation step of
catalysis(11, 12, 13, 14) .
Furthermore, the vaccinia enzyme cleaves DNA at a unique recognition
sequence(15, 16, 17) , while the cellular
enzymes, although they will cleave at specific
sequences(18, 19) , have only a limited sequence
preference(14, 20, 21, 22, 23, 24, 25) .
terminus and the linker domain are
extremely sensitive to proteolysis. We also find that noncovalent
binding of duplex DNA results in protection of the linker domain, but
not the NH-terminal domain, from proteolysis. When the
enzyme is permanently trapped in a covalent complex with a
single-stranded oligonucleotide, the linker region is rendered
sensitive to proteolysis whether duplex DNA is present or not. Taken
together, our results provide the first structural model for human Topo
I and provide important insights into the nature of its interaction
with DNA. A comparison of this model with that recently proposed for
the vaccinia viral Topo I suggests that the cellular and viral enzymes
belong to different Topo I families.
General
The recombinant proteins including (i)
the wild type and Y723F forms of full-length human Topo I (91 kDa),
(ii) the wild type and Y723F forms of the NH-terminally
deleted Topo70, which starts with an engineered methionine immediately
adjacent to residue Lys (70 kDa), and (iii) the core
fragment Topo58, which has the same NH
terminus as Topo70
but is terminated following amino acid Ala (58 kDa) were
generated and purified as described in the accompanying paper (see Fig. 1B in (5) ). Long term storage of crude
insect cell nuclear extracts containing recombinant full-length Topo I
resulted in proteolytic breakdown of the full-length protein into
fragments of 70 and 75 kDa, designated f-Topo70 and
f-Topo75(5) . Both fragments were purified and demonstrated to
be NH
-terminally deleted forms of Topo I, where f-Topo75 is
missing the first 137 residues and f-Topo70 is missing the first 174
residues(5) . Plasmid relaxation assays, SDS-PAGE,
autoradiography, and amino-terminal sequence analyses were carried out
as described in the accompanying paper(5) .
-lactoglobulin (18.4
kDa), and lysozyme (14.3 kDa). B, after the addition of PMSF,
3-µl aliquots of samples 2, 5, and 6 (displayed in panel A) were mixed with 100 µl of
dilution buffer (10 mM Tris-hydrochloride, pH 7.5, 1 mM DTT, 1 mM EDTA, 0.1 mg/ml bovine serum albumin), serially
diluted 3-fold in dilution buffer, and then subjected to plasmid
relaxation assays. The left panel shows the assays of Topo I
that was incubated with no subtilisin (sample 6). The first
two lanes of the middle panel contained a sample of the input
plasmid and a 1-kilobase pair ladder (Life Technologies, Inc.),
respectively. The remaining lanes of the middle panel show the assays of Topo I that was digested with 0.025 µg of
subtilisin (sample 5). The left panel shows the
assays of Topo I that was digested with 0.2 µg of subtilsin (sample 2). For each assay, the 3-fold dilutions are displayed
from left to right, starting at 30 ng/µl of
input Topo I. Arrows above the panels are pointed in
the direction of increasing dilution.
Antiserum and Immunoblotting
Polyclonal anti-Topo
I serum was raised in a rabbit that had been injected with purified
recombinant human Topo I. Immunoaffinity purification of anti-Topo I
serum was achieved by incubation of the crude serum with Affi-10
agarose beads (Bio-Rad) to which Topo I had been conjugated. The
affinity beads were washed extensively with 100 mM and 10
mM Tris-hydrochloride, pH 8.0, and eluted with 100 mM glycine, pH 3.0(26) . The eluate was neutralized with 0.1
volume of 1 M Tris-hydrochloride, pH 8.0, dialyzed against 50%
glycerol in phosphate-buffered saline, and stored at -20 °C.
The affinity-purified antiserum was used for chemiluminescent
immunoblotting as described previously(27) .Purification and Radiolabeling of
Oligonucleotides
DNA oligonucleotides were synthesized by DNA
Express (Fort Collins, CO), and obtained while still covalently
attached to the controlled pore glass (CPG) beads with the trityl group
on. The oligonucleotides were uncoupled from the CPG beads during an
overnight incubation in 1 ml of concentrated NH
OH at 55
°C in a screw cap tube. The tubes were cooled to -20 °C
before being uncapped, at which point the CPG beads plus
NHOH were taken up in a syringe that contained 5 ml of 80
mM ammonium acetate, pH 10.0. The diluted material was
filtered through a 0.45-µm Elutip (Schleicher & Schuell)
syringe filter before being loaded onto a POROS R2-20 column
(4.6/100-mm, Perseptive Biosystems). The reverse phase R2 column was
eluted with a 0-30% acetonitrile (ACN) gradient in 80 mM ammonium acetate, pH 10.0. The trityl-off failure (n minus-mer) sequences were found to elute at 8% ACN, while the
full-length (n-mer) oligonucleotides eluted at 20% ACN. The
20% ACN oligonucleotide pool was diluted with 1.5 volumes of 2%
trifluoroacetic acid immediately prior to being loaded onto a strong
anion exchange POROS HQ-20 column (4.6/100-mm) that was equilibrated
with HQ buffer (25 mM NaOH, 1 mM EDTA). The column
was washed with 20 ml of HQ buffer and eluted with a 0-600 mM NaCl gradient in HQ buffer. The peak fractions were pooled and
loaded back onto the R2 column, which was washed with 20 ml of 80
mM ammonium acetate, pH 10.0, to remove all traces of NaCl.
The R2 column was step-eluted with 100% ACN, and fractions were
carefully collected in order to minimize the quantity of ammonium
acetate carried over into the ACN eluate. The oligonucleotides were
dried down under vacuum, resuspended in water, and stored at -20
°C.-
P]ATP (3000 Ci/mmol) and 10 units of T4
polynucleotide kinase (Biolabs) in 100 µl of kinase buffer
(Biolabs) for 30 min at 37 °C. The reactions were terminated by
incubation at 65 °C for 10 min. Unincorporated label was separated
from the oligonucleotide by chromatography through a 1.5-ml Sephadex
G-25 spin column that was equilibrated with STE (20 mM Tris-hydrochloride, pH 7.5, 100 mM NaCl, 1 mM EDTA).
Proteolysis
Trypsin and subtilisin were purchased
from Boehringer Mannheim, and resorufin-labeled casein was purchased
from Calbiochem. All experimental digestions were carried out for 20
min at room temperature in either the standard digestion buffer (SDB)
(100 mM KCl, 10 mM Tris-hydrochloride, pH7.5, 1
mM EDTA, 1 mM DTT) or KPO digestion
buffer (KDB) (170 mM KPO, pH 7.4, 1 mM EDTA, 1 mM DTT, 10 mM MgCl), and
terminated by the addition of phenylmethylsulfonyl fluoride (PMSF) to a
final concentration of 1 mM. To assess the effect of DNA on
the activity of trypsin and subtilisin, resorufin-casein was subjected
to digestion in the presence and absence of DNA. Digestions were
carried out in SDB with or without 10 mM MgCl and
in the presence or absence of 80 µg/ml of plasmid DNA. The
reactions were terminated, and the undigested protein was precipitated
by the addition of 3 volumes of 5% trichloroacetic acid. The samples
were stored at 4 °C for 16 h, and the precipitate was removed by
centrifugation for 10 min in the microfuge. The supernatants were
neutralized by the addition of 6 volumes of 0.5 M Tris-hydrochloride, pH 8.5, and subjected to spectrofluorometric
analysis with an excitation wavelength of 574 nm and a detector
wavelength of 584 nm. In this way, proteolysis of the fluorescent
substrate was assessed by measuring the quantity of trichloroacetic
acid-soluble chromophore following digestion. This assay demonstrated
that DNA has little if any effect on the activity of either trypsin or
subtilisin in either the presence or absence of Mg (data not shown).
Purification of Radiolabeled Topo I-Oligonucleotide
Covalent Complexes
Radiolabeled Topo I-oligonucleotide complexes
were generated by incubating 40 µg of topoisomerase (full-length
Topo I or Topo70) with 5 µg of 5`-P-end-labeled
suicide oligonucleotide (either 5`-AAAAAGACTTAGAAAAATTTTT-3` or
5`-GAAAAAAGACTTAGAAAAATTTTTA-3`), in 100 µl of suicide buffer (100
mM NaCl, 10 mM Tris-hydrochloride, pH 7.5, 1 mM DTT, 1 mM EDTA, 10 mM MgCl
) for 3 h
at room temperature. The unreacted protein and protein-DNA complexes
were separated from excess suicide substrate by small scale
SP-Sepharose (Pharmacia Biotech Inc.) chromatography. The reaction
mixture was passed over a 0.25-ml bed volume of SP-Sepharose that was
equilibrated with 100 mM KPO, pH 7.4, 1 mM EDTA, 1 mM DTT. The column was washed with 20 volumes of
the same buffer to remove excess oligonucleotide and then eluted with
two 0.25-ml washes of 500 mM KPO, pH 7.4, 1 mM EDTA, 1 mM DTT. The eluate, which contained a mixture of
suicided complexes and unreacted enzyme, was stored at 4 °C.
Limited Proteolysis of Topo I
To examine the
domain structure of Topo I, we subjected recombinant full-length Topo I
to limited proteolytic digestion with a variety of proteases.
Subtilisin and trypsin were found to produce the most informative
digestion patterns (Fig. 1A). At low concentrations,
subtilisin cleaves Topo I into fragments of approximately 75 and 73 kDa (lane 5), while trypsin cleaves Topo I into fragments of sizes
between 73 and 67 kDa (lane 10). At higher concentrations,
subtilisin and trypsin generate 60- and
55-kDa fragments,
respectively, defined as s-Topo60 (lanes 2-4) and
t-Topo55 (lanes 7-9). At still higher concentrations,
fragments of
33 and
12 kDa are generated by both proteases (lanes 2 and 7).
-terminal sequence analysis. The NH terminus of s-Topo60 was found to be Lys, while
t-Topo55 begins with Lys
. Given the relative sizes of
these two proteins, the specificity of trypsin to cleave at lysine and
arginine residues, and the NH
-terminal sequence
information, the COOH termini of both fragments are predicted to be
located at or near Lys, and therefore both fragments lack
the conserved COOH-terminal
10-kDa domain.
3-fold drop in enzyme
activity. Since it has been shown previously that the smallest
NH
-terminally deleted form of Topo I that retains activity
is 63 kDa (starting at residue 231)(9) , this result suggested
that s-Topo60 (which lacks the COOH-terminal domain) may act in
conjunction with a COOH-terminal fragment to achieve activity.Identification of COOH-terminal Proteolytic
Fragments
To determine the fate of the COOH-terminal domain
during digestion, we radiolabled the COOH terminus of recombinant Topo
I by incubating the enzyme with a P-5`-end-labeled 22-base
``suicide'' oligonucleotide, which is capable of forming an
intermolecular 8-base pair duplex at its 3` end. Topo I cleaves one
strand of this duplex region, and concomitantly becomes covalently
linked to the 3` end at the site of cleavage (data not shown). However,
the topoisomerization reaction cannot be completed since the 3` segment
of the broken strand falls away from the cleaved complex, leaving it
with no 5` hydroxyl to attack the phosphotyrosine bond and reverse the
reaction. This results in the formation of a stable radiolabeled
covalent complex, which was separated from excess uncleaved
oligonucleotide by SP-Sepharose chromatography. It is important to note
that the SP-Sepharose eluate contains only a small fraction of
radiolabeled molecules, while the bulk of the protein is unlabeled
since it did not undergo suicidal cleavage. This mixture was then
digested with increasing quantities of either subtilisin or trypsin.
Fragments containing the COOH-terminal domain were identified by
autoradiography following fractionation by SDS-PAGE (Fig. 2).
This analysis revealed that the 67-75-kDa fragments each contain
the COOH-terminal domain (subtilisin lanes 5-8, trypsin lanes 12-16; compare silver stained gels in panel A with the autoradiographs in panel B). As expected,
P label is absent from the s-Topo60 and t-Topo55
fragments, confirming that they are missing the COOH-terminal domain.
It can also be seen that with increasing levels of protease, a
P-labeled COOH-terminal fragment of
15 kDa was
released from the larger fragments. This
P-labeled
COOH-terminal fragment is composed of a
5-kDa oligonucleotide that
is covalently attached to a COOH-terminal fragment of
10 kDa. (
)These results indicate that cleavage of the
67-75-kDa fragments generates a 10-kDa COOH-terminal
fragment and the s-Topo60 and t-Topo55 fragments. Though not obvious in
the figure, when the autoradiographs are superimposed on their
respective silver-stained gels, it can be seen that the radiolabeled
molecules, which are only a fraction of the total, migrate slightly
slower than the bulk of the unlabeled proteins due to the attached
oligonucleotide.
P-labeled Topo I-oligonucleotide covalent complexes.
Full-length Topo I (F.L. topo I) was allowed to undergo
suicide cleavage with a
P-5`-end-labeled 22-mer
(5`-AAAAAGACTTAGAAAAATTTTT-3`) substrate. The protein was separated
from excess 22-mer by SP-Sepharose chromatography (``Experimental
Procedures''), diluted into KDB, and digested with 2-fold
decreasing (starting at 50 ng/µl) quantities of subtilisin (lanes 2-8) or trypsin (lanes 10-16). The
samples were analyzed by 9-17% SDS-PAGE, followed by silver
staining (A) and autoradiography (B). Lane 1 contained radiolabeled suicided recombinant Topo70 that was
adjusted to 1 mM PMSF prior (PMSF first) to the addition of 50
ng/µl subtilisin. Lanes 9 and 17 contained
radiolabeled suicided Topo I that was incubated under digestion
conditions without added protease. The positions of the various
cleavage products and the proteases are indicated along the two sides
of the figure.
The Effects of Plasmid DNA on Proteolysis of Topo
I
Together the data presented thus far indicate that the actual
domain structure of Topo I closely parallels that predicted from
sequence comparisons (see Fig. 6). With this picture in mind, we
next sought to gain insight into the domain(s) of Topo I that either
interact with DNA or undergo a conformational shift upon binding to
DNA. To this end, we compared the subtilisin digestion pattern of
recombinant Topo I in the presence and absence of plasmid DNA with the
goal of identifying regions of Topo I that might either become more
resistant or more sensitive to the protease when DNA was present. After
performing preliminary experiments to ensure that DNA does not act as
general inhibitor of subtilisin (see ``Experimental
Procedures''), we subjected both wild type and Y723F mutant
full-length Topo I to digestion with increasing quantities of
subtilisin in the presence or absence of plasmid DNA (Fig. 3).
The DNA:protein mass ratio was set at 2:1 to ensure that protein-DNA
complexes remained soluble (data not shown).
-terminal domain (residues 1-197) is largely if not
entirely unfolded and is dispensable for activity. The core domain
(residues 198-651) is largely resistant to proteolysis, as is the
COOH-terminal 10-kDa domain (residues 697-765). The linker region
(residues 652-696) is sensitive to proteolysis in the absence of
DNA, but in the presence of DNA it is 10-fold more resistant to
proteolysis. In the suicided covalent complex, this same linker region
is sensitive to proteolysis both in the absence and in the presence of
excess duplex DNA. Arrows, sites of proteolytic cleavage
within the NH-terminal domain that have been defined by
amino-terminal sequencing. Broken arrows, predicted
approximate sites of limited subtilisin and trypsin cleavage within the
linker domain. Small arrow, a subtilisin cleavage site located
somewhere close to the middle of the core domain (see Fig. 4). Filled circles, putative nuclear localization signals. Open circle, a single nuclear localization signal that is
sufficient for nuclear transport.
on subtilisin digestion of Topo I. Wild type (top panel) or Y723F active site mutant (bottom
panel) Topo I proteins were digested in SDB, in the presence or
absence of 100 µg/ml of plasmid DNA (+DNA, lanes
6-10 and lanes 16-20; -DNA, lanes 1-5 and lanes 11-15), with or
without 10 mM MgCl
(+Mg, lanes 11-20; -Mg
, lanes
1-10). The 100-µl reactions contained 5 µg of Topo I
plus the following quantities of protease: 0.5 µg (lanes 1, 6,
11, and 16), 0.25 µg (lanes 2, 7, 12, and 17), 0.125
µg (lanes 3, 8, 13, and 18), 0.0625 µg (lanes 4,
9, 14, and 19), or no protease (lanes 5, 10, 15, and 20).
The samples were fractionated by 5-17% SDS-PAGE and visualized by
Coomassie Blue staining. Lane 21 contained the molecular mass
markers (Sigma) bovine serum albumin (66 kDa), ovalbumin (45 kDa),
trypsinogen (24 kDa),
-lactoglobulin (18.4 kDa), and lysozyme
(14.3 kDa).
. The
digests were performed either in the absence or presence of a 22-base
pair duplex oligonucleotide DNA (-DNA, lanes 2-9,
+DNA, lanes 11-18). To ensure that protein DNA
complexes were soluble (data not shown), the DNA:topoisomerase mass
ratio was set at 2:1 (2 µg of duplex 22-mer and 1 µg of enzyme
in a 30-µl reaction). The digestion products were fractionated by
9% SDS-PAGE and visualized by silver staining. Lanes 1, 10, and
19, respectively, contained 1 µg of untreated full-length Topo
I Y723F, Topo70 Y732F, and Topo58. Lanes 2 and 11 contained
samples that were incubated under digestion conditions without any
subtilisin (NO Subt.). B, Topo70 was digested under
the same conditions described above for full-length Topo I. C,
the duplex 22-mer used in the above experiments contains the
hexadecameric sequence (underlined), which is known to be a
high affinity binding site for mammalian
topoisomerases(19, 35, 36) . The site of Topo
I cleavage is indicated with a small
arrow.
, the full-length protein
was nearly completely converted into a combination of 73- and 75-kDa
fragments at low subtilisin concentrations (Fig. 3; lanes
4, 9, 14, and 19). Thus, the initial
removal of the NH
-terminal domain from the full-length
protein is relatively unaffected by the presence of DNA. In contrast,
proteolysis of the 73-kDa fragment to produce the s-Topo60 core is
inhibited approximately 10-fold in the presence of DNA (for example,
compare lanes 1-4 with lanes 6-9 of both panels A and B). Thus, cleavage between the s-Topo60
core domain and the 10-kDa COOH-terminal domain is inhibited by
bound DNA. The block to proteolysis within this region was of the same
magnitude in the presence or absence of Mg
(compare lanes 1-10 with lanes 11-20 of both panels A and B) and was observed with wild type and
active site mutant enzymes. In the case of the wild type enzyme, the
plasmid DNA is relaxed to completion before subtilisin is added.
However, the Y723F protein contains less than 0.1 units (one unit is
the amount of enzyme required to fully relax 1 µg of plasmid DNA in
10 min at 37 °C) of endogenous insect cell topoisomerase/µg of
recombinant enzyme. Therefore, the 20-min room temperature incubation
with 5 µg of mutant enzyme results in very little relaxation of the
10 µg of plasmid DNA present during digestion. Thus, supercoiled
(when the Y723F mutant is used) and relaxed plasmid DNAs (when the wild
type enzyme is used) are equally effective at blocking cleavage between
the s-Topo60 core and the
10-kDa COOH-terminal domains.
The Effect of Duplex Oligonucleotides on Proteolysis of
Full-length Topo I and Topo70
There is mounting evidence that
Topo I has a preference for binding to DNA nodes, points at which two
duplexes cross over(29, 30) . Thus, it is possible
that node binding causes resistance to proteolysis in the linker region
between the core and COOH-terminal domains. To test this possibility,
we examined the effect of a short duplex oligonucleotide on the
kinetics of limited proteolysis. The rationale for this experiment lies
in the fact that, while plasmid DNA molecules, and particularly
supercoiled plasmid DNA, have a propensity to form intramolecular
nodes(31) , short oligonucleotides do not. In the same
experiment, we also sought to examine the effect of removing the
NH-terminal domain on the pattern of limited proteolysis.
Full-length Topo I and the NH-terminally truncated Topo70
were subjected to digestion with increasing concentrations of
subtilisin in the presence or absence of a 10-fold molar excess of a
22-base pair duplex oligonucleotide (Fig. 4). The
oligonucleotide (Fig. 4C) contained the hexadecameric
sequence from the rDNA of Tetrahymena plus upstream and
downstream base pairs that are known to be important for maximal Topo I
cleavage of this high affinity binding
site(18, 19, 32, 33, 34, 35, 36) .
The results demonstrate that, as with the plasmid DNA, cleavage of
full-length Topo I to generate the 73-kDa fragment was only
slightly inhibited (<2-fold) by the presence of the duplex
oligonucleotide (compare lanes 3-6 to lanes
12-15 of panel A). In contrast, the oligonucleotide
inhibited by
10-fold the cleavage of both Topo70 and the
73-kDa fragment that is generated by proteolytic removal of the
amino terminus from full-length Topo I (compare lanes 3-9 to lanes 12-18 of panels A and B).
73-kDa
fragment and Topo70 were very similar (Fig. 4; compare lanes
5-9 and lanes 16-18 of panels A and B). Like the full-length protein, Topo70 appeared to be
cleaved by subtilisin at or near Lys
, since a
58-kDa
(s-Topo58) fragment that migrated in SDS-PAGE slightly faster than the
recombinant Topo58 was produced. Taken together, these results
demonstrate that the lack of the NH
-terminal domain does
not affect the digestion pattern of Topo I. It can also be concluded
that a short duplex oligonucleotide inhibits cleavage between the core
and the COOH-terminal domains to approximately the same extent as long
plasmid DNA.33 kDa (designated s-Topo33; Fig. 4A, lanes 8 and 9). Similarly,
further digestion of s-Topo58 produces a fragment of
30 kDa, which
migrates slightly slower than subtilisin (designated s-Topo30; Fig. 4B, lanes 8 and 9). From
amino-terminal sequence analysis, we know that the s-Topo60 core
fragment from full-length Topo I starts at residue Lys
as
compared with the Lys
start of Topo70. Therefore, we
predict that the s-Topo33 and s-Topo30 fragments, respectively, start
at residues Lys
and Lys
and terminate at or
near the same residue somewhere within the middle of the core domain.
Thus, limited proteolysis can also be used to subdivide the core domain
into two globular segments of roughly equal size.
Proteolysis of the Suicided Covalent Complex Is Not
Affected by DNA
To further examine the effect of DNA on limited
proteolysis, we determined the sensitivity to proteolysis of enzyme
molecules that were permanently trapped in the covalent complex with
DNA. We generated radiolabeled Topo70 covalent complexes as described
above (Fig. 2) and subjected them to digestion with increasing
quantities of subtilisin in the presence or absence of plasmid DNA. The
digestion products were then fractionated by SDS-PAGE and visualized by
immunoblotting (Fig. 5B) and autoradiography (Fig. 5A). The immunoblot displays the digestion pattern of
the bulk unsuicided protein and confirms that Topo70 is 10-fold
more resistant to cleavage when plasmid DNA is present (panel
B, compare -DNA lanes 1-8, and +DNA
lanes 11-18). In contrast, the autoradiograph reveals that
the radiolabeled Topo70-oligo complexes are digested with equal
efficiencies in the presence or absence of plasmid DNA (panel
A, compare -DNA lanes 1-8 and +DNA
lanes 11-18). A comparison of the Topo70 immunoblot signals
to those of the autoradiograph reveals that subtilisin cleaves the
suicided Topo70 with an efficiency that closely parallels cleavage of
the bulk unlabeled Topo70 in the absence of DNA. This result suggests
that plasmid DNA is unable to inhibit proteolysis of the suicided
molecules and that a covalent Topo70 single-stranded oligo complex is
just as sensitive to proteolysis as Topo70 in the absence of plasmid
DNA. This result is highly reproducible and has been obtained using the
full-length Topo I and using trypsin as the protease instead of
subtilisin (data not shown).
P-5` end-labeled 25-mer (5`-GAAAAAAGACTTAGAAAAATTTTTA-3`)
suicide substrate and then fractionated by SP-Sepharose chromatography
to remove the excess 25-mer (``Experimental Procedures'').
The protein was then digested in KDB with 2-fold increasing quantities
of subtilisin (starting at 50 ng/µl), in the presence or absence of
66 µg/ml plasmid DNA (+DNA, lanes
11-20; -DNA, lanes 1-10).
Reactions were terminated by sequentially adjusting to 0.1 mM PMSF, 1 M NaCl, and then 0.1% SDS, before being boiled.
Half of each sample was fractionated by 9-17% SDS-PAGE and
analyzed by autoradiography (panel A, Autoradiograph). The other half was fractionated by
9-17% SDS-PAGE and analyzed by immunoblotting with
affinity-purified anti-Topo I serum (panel B, Immunoblot). Lanes 9 and 19 contained
samples that were incubated in the absence of subtilisin (NO
subt.). Lanes 10 and 20 contained samples that
were adjusted to 0.1 mM PMSF prior (PMSF first) to the
addition of the highest concentration of subtilisin used. Lanes 10 and 20 are not labeled on the immunoblot in panel
B, since the PMSF control samples were not included in this
analysis.
Domain Organization of Human Topo I
Limited
digestion with subtilisin and trypsin reveals that human Topo I is
comprised of four major domains whose boundaries closely parallel those
predicted from sequence comparisons of cellular Topo I enzymes (Fig. 6). The 24-kDa unconserved NH-terminal domain
(residues Met-Lys) is highly sensitive
to proteolysis and is largely if not completely unfolded (see
accompanying paper(5) ). It is cleaved at the carboxy side of
residues Lys
and Lys
by an unidentified
insect cell protease and at residues Lys
and Lys
by subtilisin and trypsin, respectively. The
NH
-terminal domain is followed by the conserved 54-kDa
core domain (residues Glu
-Ile
), which
is connected to the 8-kDa COOH-terminal domain (residues
Gln
-Phe
) via a short linker region.
In the absence of DNA, the linker region is sensitive to proteolysis
and is cleaved in at least two positions, probably located at
Lys
and Lys
.
DNA Binding
Proteolysis of the linker segment
located between the core and COOH-terminal domains is inhibited by
either plasmid DNA or a 22-base pair duplex oligonucleotide. Since the
length of DNA was not a factor in protecting the linker domain from
proteolysis, we conclude that binding to DNA nodes, such as those
formed when two duplexes cross within a supercoiled
plasmid(29, 30, 31) , is probably not
responsible for protection of the linker domain. However, it is
difficult to exclude the possibility that the enzyme binds two segments
of a plasmid or two separate duplex oligonucleotides to form the
equivalent of a node within the binding pocket of the enzyme. The
presence or absence of Mg was found to have no effect
on the patterns of limited proteolysis of either the free or DNA bound
protein, suggesting that Mg
does not have any major
effect on enzyme structure. An examination of the Y723F active site
mutant enzyme revealed that the DNA-induced protection of the linker
segment occurs in the absence of DNA strand breakage, when Topo I is
noncovalently bound to DNA. In contrast, the linker domain of
permanently trapped covalent complexes is sensitive to proteolysis
whether or not DNA is present.
-Glu
) suggests that it
may bind directly to DNA. Thus, the first model would most easily
explain our observation that the linker domain resists proteolysis when
Topo I is noncovalently bound to duplex DNA. However, other than being
positively charged, the linker region is not well conserved among the
cellular topoisomerases. Therefore, if it is involved in direct DNA
contacts, one would have to suppose that the interactions would be
somewhat flexible in their amino acid sequence requirements.
Comparison with the Vaccinia Viral Topo I
Limited
proteolysis studies of the vaccinia virus Topo I have revealed that the
enzyme has a tripartite domain organization, being comprised of a 9-kDa
NH-terminal domain, an 8-kDa central domain, and a 20-kDa
COOH-terminal domain, which contains the active site
tyrosine(4, 28) . The three domains are connected via
protease-sensitive linker regions. Hence, the limited tryptic digestion
pattern of the vaccinia enzyme generates fragments of 9, 8, and 20 kDa.
This pattern differs substantially from the tryptic digestion pattern
of the human enzyme. For example, the COOH-terminal domain of the human
enzyme is only 10 kDa in size and is connected to a core domain of
54 kDa via a short proteolytically sensitive linker domain.
Furthermore, proteolytic cleavage of the vaccinia enzyme within either
of the two linker regions results in loss of activity(4) . In
contrast, cleavage within the linker domain of the human enzyme has
little effect on Topo I activity. Relative to this observation, we have
recently found that Topo I activity can be reconstituted by mixing
individually expressed core and COOH-terminal domains, the latter of
which is inactive by itself.
-terminal
domain. Furthermore, when the human enzyme is covalently bound to a
single-stranded substrate, the linker region is rendered sensitive to
proteolysis regardless of the presence or absence of duplex DNA. The
vaccinia enzyme has only 43% sequence similarity and 20% sequence
identity with the human enzyme(28) . Thus, even
though the viral and cellular enzymes display very similar catalytic
and biochemical properties, the weak sequence similarity between the
two and the differences in domain organization as assayed by limited
proteolysis strongly suggest that the cellular and viral topoisomerases
comprise separate families of type I enzymes. More detailed structural
and/or crystallographic analyses of the two proteins will be required
to confirm or disprove this prediction.
)-terminal truncation of Topo I,
missing the first 174 amino acids; Topo58, COOH-terminal truncation of
Topo70, missing the last 106 amino acids; Y723F, Topo I with active
site tyrosine at position 723 replaced with phenylalanine; PMSF,
phenylmethylsulfonyl fluoride; CD, circular dichroism; DTT,
dithiothreitol; CPG, controlled pore glass; ACN, acetonitrile; SDS,
sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; SDB,
standard digestion buffer; KDB, potassium phosphate digestion buffer.))
We thank Leon Parker, Knut Madden, Xiayang Qiu, Sam
Whiting, and Sharon Schultz for helpful comments and valuable
discussions during preparation of the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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