(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-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.
Eukaryotic topoisomerase I (Topo I) ()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.
Sequence comparisons of the
cellular eukaryotic Topo I enzymes ()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) .
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-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) .
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 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.
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), -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.
The purified oligonucleotides were radiolabeled by
incubating 5 µg of oligonucleotide with 100 µCi of
[-
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).
To learn more about the amino
acid sequence content of the Topo I fragments, we subjected s-Topo60
and t-Topo55 to NH-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.
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 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.
Figure 2:
Subtilisin and trypsin digestion of 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.
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-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.
Figure 3:
The effect of plasmid DNA and
Mg 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).
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. 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.
In the presence or
absence of either DNA or Mg, 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.
In general, the digestion patterns of the 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.
It should be noted that with high concentrations of
subtilisin, the s-Topo60 core fragment is further digested into at
least one fragment of 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.
Figure 5:
Subtilisin digestion of suicided Topo70 in
the presence or absence of plasmid DNA. Topo70 was incubated with a 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.
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-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.
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-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.