From the Department of Molecular and Structural Biology, University
of Aarhus, C.F. Møllers Allé, Building 130, DK-8000, Aarhus C,
Denmark, ¶ Medizinische Poliklinik, University of Würzburg
Medical School, Klinikstrasse 6-8, Würzburg D-97070, Germany, and
Section of Molecular Genetics and Microbiology, University of
Texas at Austin, Texas 78712
Received for publication, December 6, 2000, and in revised form, March 1, 2001
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ABSTRACT |
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All eukaryotic forms of DNA topoisomerase I
contain an extensive and highly charged N-terminal domain. This domain
contains several nuclear localization sequences and is essential for
in vivo function of the enzyme. However, so far no direct
function of the N-terminal domain in the in vitro
topoisomerase I reaction has been reported. In this study we have
compared the in vitro activities of a truncated form of
human topoisomerase I lacking amino acids 1-206 (p67) with the
full-length enzyme (p91). Using these enzyme forms, we have identified
for the first time a direct role of residues within the N-terminal
domain in modulating topoisomerase I catalysis, as revealed by
significant differences between p67 and p91 in DNA binding, cleavage,
strand rotation, and ligation. A comparison with previously published
studies showing no effect of deleting the first 174 or 190 amino acids
of topoisomerase I (Stewart, L., Ireton, G. C., and Champoux,
J. J. (1999) J. Biol. Chem. 274, 32950-32960;
Bronstein, I. B., Wynne-Jones, A., Sukhanova, A., Fleury, F.,
Ianoul, A., Holden, J. A., Alix, A. J., Dodson, G. G.,
Jardillier, J. C., Nabiev, I., and Wilkinson, A. J. (1999) Anticancer Res. 19, 317-327) suggests a pivotal role of
amino acids 191-206 in catalysis. Taken together the presented data indicate that at least part(s) of the N-terminal domain regulate(s) enzyme/DNA dynamics during relaxation most probably by controlling non-covalent DNA binding downstream of the cleavage site either directly or by coordinating DNA contacts by other parts of the enzyme.
Eukaryotic topoisomerase I (topo
I)1 is a monomeric enzyme
that plays a major role in important cellular processes by regulating the topology of DNA. The enzyme relaxes negative and positive supercoils arising as a consequence of DNA processes such as DNA transcription, replication, recombination, and chromosome condensation (1).
Mechanistically, topo I acts by introducing transient single-strand
breaks into the DNA double helix. The catalytic cycle can be subdivided
into several steps including: (i) non-covalent DNA binding, (ii)
cleavage, (iii) strand rotation, (iv) religation, and (v) enzyme
turnover. The cleavage and religation events constitute two reverse
phosphoryl transfer (transesterification) reactions. During the
cleavage reaction, an active-site tyrosine residue of the enzyme is
used as a nucleophile to break a phosphodiester bond of the DNA
backbone, generating a covalent enzyme-(3'-phosphotyrosyl)-DNA linkage
and a free 5'-hydroxyl group (2-4). This 5'-hydroxyl group provides
the nucleophile for the religation reaction that restores intact DNA.
The solved crystal structure of an N-terminal-truncated version of the
human topo I together with proteolytic analyses show that the enzyme is
organized into four structural domains. These consist of an N-terminal
domain (amino acids 1-206), a core domain (amino acids 207-635), a
linker domain (amino acids 636-712), and a C-terminal domain (amino
acids 713-765) (2, 3). The C-terminal domain contains the active-site
tyrosine (Tyr723), which together with the catalytic
residues Arg488, Lys532, Arg590,
and His632 of the core domain constitutes the active site
of the enzyme (4-8). Structural data show that the core and C-terminal
domains form a clamp structure that wraps around the DNA and, together with the helix-turn-helix linker domain (1), contacts DNA in a region
extending 4 base pairs upstream and 9 base pairs downstream of the
cleavage site (3). Based on this structural information of the human
topo I-DNA complex, a model for strand rotation (topoisomerization) has
been proposed. According to this "controlled rotation" model, rotation of the cleaved strand around the intact strand is partially hindered by contacts between the rotating DNA and part of the core and
linker domains (3). The involvement of the linker in controlling strand
rotation has recently been supported by biochemical studies showing
that the sensitivity of topo I toward camptothecin in relaxation
depends on a functional linker domain (9). The recently published
crystal structure of a topo I form encompassing a few residues of the
N-terminal domain (amino acids 203-214) demonstrates an interaction
between Trp205 and the hinge region of the core domain
(37). This interaction has been proposed to be important for the
flexibility of the enzyme clamp, allowing controlled strand rotation.
Beside such speculations, the putative enzymatic function(s) of the
N-terminal domain of topo I has remained unknown. Based on biochemical
analyses, it is assumed to be largely unfolded or highly dynamic (2,
10). It is essential for nuclear localization of topo I, and four
nuclear localization signals have been identified within the domain
(11). For years it has been considered unimportant for catalysis, since the first purified forms of catalytically active topo I were in fact
proteolytic degradation products lacking this domain (12, 13). The
apparent dispensability of the N-terminal domain was recently supported
by deletion studies showing that human topo I variants lacking either
the first 174 (9) or the first 190 amino acids (14) show no obvious
defects in vitro.
In the present work we have addressed the possible role of the
N-terminal domain in topo I catalysis by comparing the in
vitro activities of a truncated version of human topo I lacking
amino acids 1-206 (p67) with those of the full-length enzyme (p91). We
have found significant differences between p67 and p91 in DNA binding,
cleavage, strand rotation, and ligation. The sum of our results held
together with previous reports showing no effect of deleting the first
190 amino acids of the enzyme (14) suggests a particularly important
role of amino acids 191-206 of the N-terminal domain in catalysis most
probably by coordinating non-covalent enzyme-DNA interactions during
the individual steps of topoisomerization.
Reagents and Enzymes--
Me2SO (ACS grade),
phenylmethylsulfonyl fluoride, cytochrome c, dApdG, and
camptothecin were from Sigma. Camptothecin was dissolved in 50% (v/v)
Me2SO at 600 µM and stored at Yeast Strains and Construction of Human TOP1 Expression
Plasmids--
The Saccharomyces cerevisiae top1 null strain
RS190 (15) was kindly provided by R. Sternglanz (State University of
New York, Stony Brook, NY). Plasmid pHT143, for expression of
recombinant full-length human topo I (p91) in S. cerevisiae,
was described previously (16). Plasmid pHT148, for expression of a
fusion of GST to the N terminus of amino acids 207-765 of human topo I
(GST-p67), was constructed by first cloning a polymerase chain reaction
fragment of pGEX-2TK containing the GST tag and the polylinker into a
BamHI/EcoRI fragment of pHT143. The original
BamHI site in pHT143 was destroyed, and the topo I gene was
deleted from pHT143 by this cloning step, generating a new cloning
vector referred to as pRS426-GAL-GST. Subsequently, a polymerase chain
reaction fragment containing amino acids 207-765 of human topo I was
cloned into a BamHI/EcoRI fragment of
pRS426-GAL-GST, generating pHT148. pHT147, for expression of a
GST-tagged N-terminal fragment (p25) of human topo I, was constructed
by inserting a polymerase chain reaction fragment encoding amino acids
1-218 of topo I into the BamHI/EcoRI site of the
pGEX-2TK vector. In the pHT148 and pHT147 constructs, the GST tag and
the topo I fragments were separated by specific cleavage at a thrombin
protease site.
Expression and Purification of Recombinant Forms of Human
Topo I--
The plasmids pHT143 and pHT148 for expression of p91 and
GST-p67, respectively, were transformed into the yeast S. cerevisiae strain RS190. Crude cell extracts from 12 liters of
yeast culture expressing the p91 or GST-p67 were prepared, and the
proteins were purified on two heparin-Sepharose columns and a
phenyl-Sepharose column essentially as described previously (17). For
purification of p67, the GST tag was cleaved off by thrombin
before applying the protein preparations on the phenyl-Sepharose
column. For comparability, p91 was subjected to this procedure too but
leaving out the thrombin protease. The purified enzymes were
concentrated, and the buffers were exchanged on a 0.5-ml Source 15S
column. In the final step, p67 and p91 were eluted with a buffer
containing 600 mM NaCl, 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, and 10% glycerol. A GST-tagged N-terminal
fragment of human topo I, GST-p25, was expressed from the plasmid
pHT147 in the bacterial strain BL21 (18). Bacteria were grown at
37 °C in 200 ml of LB medium containing 0.1 mg/ml ampicillin
until A600 had reached 0.6. Expression of
GST-p25 was induced by 1 mM isopropyl
Thrombin Cleavage--
In a standard digestion, 1 mg of GST-p67
was incubated with 150 units of thrombin for 1 h at room
temperature in an 8-ml volume of 1× PBS containing 5 mM
dithiothreitol. Subsequently, the digestion mixture was diluted to 50 ml with 1× PBS and passed over a 2-ml prepacked glutathione-Sepharose
column three times to remove uncleaved fusion protein and liberated GST
tag. The N-terminal amino acid sequence of p67 after thrombin cleavage
was
Gly-Ser-Arg-Arg-Ala-Ser-Val-Gly-Ser-Pro-Glu207-Glu208,
with the start of the topo I sequence indicated in bold letters.
Synthetic DNA Substrates--
Oligonucleotides for
construction of topo I suicide substrates were synthesized on a model
394 DNA synthesizer from Applied Biosystems. The sequences and the
preparation of the two classes of suicide substrates containing a
protruding noncleaved or cleaved strand were described in detail
previously (16). The sequences for constructing the
oligonucleotides for the non-suicide DNA fragment used for the filter
binding assay were: OL100T,
5'-gctatacgaattcgctataattcatatgatagcggatccaaaaaagacttagaaaaaaaaaaagcttaagcaacatatggtatcgtcggaattcaatgag; OL100B,
5'-ctcattgaattccgacgataccatatgttgcttaagctttttttttttctaagtcttttttggatccgctatcatatgaattatagcgaattcgtatagc.
Topo I-mediated DNA Cleavage--
Standard cleavage reactions
were performed by incubating 50 fmol of suicide DNA substrate
(OL19/OL27) with 500 fmol of p91 or p67 in 20 µl of topo I reaction
buffer (10 mM Tris-HCl, pH 7.5, 5 mM
CaCl2, 5 mM MgCl2) at indicated
temperatures. The cleavage reactions were stopped by the addition of
NaCl to a final concentration of 375 mM, and cleavage
products were analyzed on an 8% SDS Tris-glycine polyacrylamide gel.
Topo I-mediated Ligation--
Active topo I-DNA cleavage
complexes containing the enzyme covalently attached at an internal
(using DNA substrate OL19/OL27) or a terminal (using DNA substrate
OL22/OL25) position were generated by preincubating 50 fmol of suicide
DNA substrate with 500 fmol of topo I at 37 °C for 5 min. This
reaction was performed as described for topo I-mediated cleavage and
terminated by the addition of 300 mM NaCl. Intramolecular
DNA ligation mediated by topo I covalently coupled at an internal
position was performed by continuing incubation in the presence of 1 µM dApdG in 50 µl of topo I reaction buffer and 300 mM NaCl. Intermolecular DNA ligation mediated by topo I
covalently attached at a terminal position was performed by incubating
the cleavage complexes in the presence of 0.02 µM
28/28-mer duplex DNA (OL32/OL33) in 50 µl of topo I buffer and 300 mM NaCl. All reactions were stopped by the addition of SDS
to a final concentration of 0.1% (w/v), and DNA was precipitated with
3 volumes of ethanol. The samples were subsequently digested with 1 mg/ml trypsin in 20 µl of 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA for 30 min at 37 °C. Finally, the samples were
analyzed on 12% denaturing polyacrylamide gels.
Assays for Topo I-mediated Relaxation of Plasmid DNA--
The
relaxation activity of human topo I was assayed for a series of enzyme
concentrations and time points as indicated. Unless otherwise stated,
reactions were performed in 20 µl of a standard relaxation buffer (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM EDTA,
5 mM MgCl2) and 50 fmol of negatively
supercoiled pBR322. Relaxation reactions were stopped by the addition
of SDS to a final concentration of 0.1% (w/v). Samples were
proteolytically digested with 0.5 µg/ml proteinase K for 30 min at
37 °C before separating the products on a 1% agarose gel with 0.5×
Tris borate electrophoresis buffer at 50 V for 12 h at 4 °C.
DNA was visualized by subsequent staining of the gel with 0.5 µg/ml
ethidium bromide and the gel image was acquired using the Bio-Rad Gel
Doc-2000 system.
DNA Binding Assay--
DNA binding of p25 and cytochrome
c was assayed by nitrocellulose filter binding essentially
as described previously (19, 20). The proteins were incubated with 20 fmol of 5'-end-labeled DNA substrate as indicated in a binding buffer
containing 50 mM Tris-HCl, pH 7.5, 0.1 mM
dithiothreitol, 5% glycerol, 1 mM EDTA, and 20 mM NaCl in a 50-µl reaction volume for 5 min at room
temperature. Subsequently, the samples were applied to a 0.2-µm
nitrocellulose filter under vacuum. To reduce nonspecific binding of
the DNA substrate, the nitrocellulose filter was pre-wetted in binding buffer. After application of the samples, the filter was washed in 500 µl of binding buffer to remove unbound DNA substrate and dried. The
percentage of input DNA retained on the filter was determined using
PhosphorImager. The data were corrected for the nonspecific binding of
free DNA, which was generally 1-2% of the total DNA.
Polyacrylamide Gel Electrophoresis--
Reaction products from
the cleavage assays were mixed with 1/10 volume of an SDS sample buffer
containing 10% glycerol (v/v), 4% SDS (w/v), 50 mM
Tris-HCl, pH 6.8, 0.01% Serva Blue G (w/v), and 200 mM
The trypsin-digested products from the ligation assays were mixed with
1 volume of 80% (v/v) deionized formamide, 50 mM Tris borate, pH 8.3,, 1 mM EDTA, 0.05% (w/v) bromphenol blue,
and 0.05% (w/v) xylene xyanol, and the mixtures were heated to
90 °C for 2 min and applied to a 12% denaturing polyacrylamide gel.
The labeled reaction products were visualized by a model SF Molecular Dynamics PhosphorImager. The amount of topo I-mediated DNA cleavage and
ligation was quantified by integrating the area under the curve for
each radioactive band using the ImageQuant software from Molecular Dynamics.
DNA Relaxation Activity of p67 and p91--
To investigate the
effects on enzymatic activity of deleting the N-terminal domain of
human topo I, recombinant p67 (amino acids 207-765) and p91 (amino
acids 1-765) were purified from yeast (see Fig.
1) and assayed for DNA relaxation
activity. Two sets of time course experiments were performed in a low
salt buffer (5 mM MgCl2) at 37 °C. In the
first set, with a 3-fold molar excess of plasmid DNA compared with
enzyme, p67 relaxed supercoiled pBR322 16-64 times faster than p91
(Fig. 2A). In the second set,
with the enzyme concentration increased to a 2-fold molar excess
compared with plasmid substrate, the relaxation rate of p67 was slower than that of p91 (compare lanes 2 and 8 in Fig.
2B). Thus, the relative activity of p67 with respect to p91
is dependent on the molar ratio between enzyme and DNA.
At a molar excess of plasmid DNA, the rate-limiting step for relaxation
of the total substrate population is the enzyme-DNA dissociation rate
(21). When changing the molar ratio between enzyme and DNA to an excess
of enzyme, the majority of DNA molecules become occupied by at least
one enzyme molecule. In consequence, enzyme-DNA dissociation is no
longer rate-limiting for relaxation. In this context, our results
suggest that deleting the N-terminal domain of human topo I increases
the dissociation rate of the enzyme to yield a higher turnover number.
This interpretation is further supported by the more distributive
relaxation mode of p67 relative to p91 (Fig. 2A, compare
lanes 2-4 with lanes 8-10), where the truncated
enzyme appears to leave its substrate after removing only a few
supercoils at a time. At the conditions used, the relaxation mode of
full-length topo I is known to be highly processive, going through
multiple rounds of relaxation before dissociating from its substrate
(22, 23). Our data with p91 are consistent with this expectation. The
relatively low relaxation activity of p67 at a molar excess of enzyme
(Fig. 2B) may be explained in two ways. Either it reflects a
slower catalysis of p67 relatively to p91 or a lower DNA affinity of p67 and a consequent reduction in the percentage of DNA-bound enzyme at
a given time.
Involvement of the Human Topo I N-terminal Domain in DNA
Binding--
The shift from a processive to a more distributive
relaxation mode of topo I upon removal of amino acids 1-206 suggests a role of the N-terminal domain (or at least part of the N-terminal domain) in non-covalent DNA binding. To evaluate this possibility, we
compared the salt sensitivity of p67 and p91 in relaxation and used
this as a measure for their respective DNA affinities. It is well known
that high salt concentration inhibits topo I-catalyzed DNA relaxation
by inhibiting DNA binding (24).
The two enzymes were incubated with supercoiled pBR322 at various
concentrations of NaCl, and the resulting products were analyzed by gel
electrophoresis. The amount of supercoiled pBR322 remaining after
incubation with topo I was quantified, and the relaxation activity was
plotted as a function of the salt concentration (Fig.
3A). The p67 topo I showed a
significantly lower salt optimum (25-75 mM NaCl) than p91
and was completely impaired in relaxation at 150 mM of
NaCl. By contrast, the relaxation optimum for p91 was in the 100-150
mM NaCl range. These results are consistent with a lowered
DNA affinity of p67. In agreement with this finding, an N-terminal
fragment of human topo I (amino acids 1-218) binds DNA in a filter
binding assay (Fig. 3B). Taken together, the obtained results suggest a role of the N-terminal domain or at least part of it
in non-covalent DNA binding.
The N-terminal Domain Is Important for Interaction with DNA
Downstream of the Cleavage Site--
For further characterization of
the N-terminal domain in the individual chemical steps of topo I
catalysis, the cleavage and ligation reactions of p67 and p91 were
compared using synthetic suicide DNA substrates (16, 25, 26). These
substrates support cleavage, whereas religation is temporarily
prevented due to dissociation of a short oligonucleotide containing the
5'-OH end generated during cleavage (Fig.
4A, right panel).
Cleavage complexes containing the enzyme covalently attached at an
internal or a terminal position can be obtained by using substrates
with strand interruptions located next to the cleavage site on either
the scissile or the non-scissile strand, respectively (16). DNA
ligation mediated by the active cleavage complexes can be initiated by
the addition of an excess of appropriate ligator strands containing
free 5'-hydroxyl ends. Cleavage complexes containing the enzyme
attached at an internal position are able to ligate single-stranded DNA
complementary to the base sequence of the noncleaved strand (referred
to as intramolecular ligation; Fig. 4B, right
panel). Intramolecular ligation can be performed with ligators as
short as 2 bases and proceeds in the presence of 1 M NaCl.
Thus, non-covalent DNA interaction is not required for intramolecular
ligation (27). Intermolecular ligation, i.e. ligation of
duplex DNA to cleavage complexes carrying topo I covalently attached at
a blunt end (Fig. 4C, right panel), is completely
abolished at 1 M salt and depends strongly on non-covalent enzyme interactions to the ligator strands.
The cleavage reactions of p67 and p91 were investigated by incubating
the enzymes with the suicide substrate OL19/OL27 at 37 °C. Fig.
4A shows the extent of covalent complex formation plotted as
a function of time. The cleavage reactions for both enzymes were quite
fast, completed within the first 40 s of incubation, and no
significant difference between the two enzymes could be observed at
these conditions. Note, however, that at low temperatures, the cleavage
rates were markedly different for p67 and p91 (see below; Fig.
7A).
To investigate intra- and intermolecular ligation, topo I was mixed
with the appropriate suicide DNA substrate to obtain active cleavage
complexes with the enzyme covalently attached at an internal (substrate
OL19/OL27) or a terminal position (substrate OL22/OL25), respectively.
After incubation for 5 min at 37 °C, the salt concentration was
increased to 300 mM to prevent further cleavage, and the
appropriate ligator strands were added (5'-HO-dApdG for intramolecular
ligation and OL32/OL33 for intermolecular ligation). Figs. 4,
B and C, show the percentage of cleavage
complexes converted to ligation products plotted as a function of the
incubation time. As shown in Fig. 4C, the ability of p67 to
mediate intermolecular ligation was severely compromised compared with
p91, whereas the effect of deleting the N-terminal domain on
intramolecular ligation was quite modest (no more than a 2-fold
reduction in the initial reaction rate of p67 compared with p91; Fig.
4B). From the data in Fig. 4C, we estimate that
the initial reaction rate for intermolecular ligation is at least
25-fold slower for p67 than for p91.
The most obvious difference between the two forms of ligation is their
different requirements for enzyme interaction to DNA downstream of the
cleavage site. During intramolecular ligation, the ligator strand can
be positioned by complementary base pairing, so that the 5'-OH group
may be oriented within the topo I active site for nucleophilic attack
on the phosphotyrosyl bond. Due to the absence of base pairing,
intermolecular ligation depends on protein-mediated positioning of the
ligator strand. Accordingly, the ligation results suggest that residues
within the N-terminal domain promote enzyme interaction to DNA
downstream of the cleavage site. Such a function would be consistent
with the lowered DNA affinity of p67 relative to p91 (Fig.
3A) and with the ability of the N-terminal domain to bind
DNA non-covalently (Fig. 3B).
Camptothecin Sensitivity of p67 and p91 in Relaxation--
The
structural and biochemical data on human topo I have led to the
proposal of the controlled rotation model to explain the topoisomerization step of catalysis. According to this model, strand
rotation is controlled by enzyme-DNA contacts downstream of the
cleavage site (1, 3). The multiple contacts of the linker domain (amino
acids 636-712) to DNA downstream of the cleavage site (1, 3) together
with the recent finding that blockage of strand rotation by
camptothecin depends on an intact linker (9) suggest that this domain
could play a role in controlling topoisomerization. In this study we
present evidence that residues of N-terminal domain, like the linker
region, are important for enzyme interaction to DNA downstream of the
cleavage site either by direct interaction(s) or by coordinating DNA
interaction(s) of other parts of the enzyme. This would suggest an
expanded version of the controlled rotation model in which residues of
the N-terminal domain along with the linker domain are involved in
controlling strand rotation. To address this possibility, the effect of
deleting the N-terminal domain on the camptothecin sensitivity of the
enzyme was investigated.
The camptothecin sensitivities of p67 and p91 in relaxation were
assayed in molar excess of enzyme over DNA to circumvent possible
effects due to a slow dissociation rate and enzyme turnover in the
presence of camptothecin. Note that camptothecin blocks religation and
will thereby slow down all the subsequent steps of topo I catalysis.
However, first it was necessary to slow down the reaction rate, which
is too fast under standard conditions employing 5 mM
MgCl2 at 37 °C (see Fig. 2B), for the
inhibition by camptothecin to be measured accurately. We found that
excluding MgCl2 from the reaction buffer slows down the
reactions of both p67 and p91 sufficiently (compare Figs.
5 and 2) (10, 26, 28-31). To obtain more
physiologically relevant conditions, 75 mM NaCl, at which
concentration the two enzymes show similar activities (see Fig. 3), was
added to the reaction mixture. Thus, the effect of camptothecin (60 µM) was assayed at 37 °C in the absence of MgCl2 and presence of 75 mM NaCl (Fig. 5). The
relaxation rate of p67 was practically unaffected by camptothecin (Fig.
5A, compare lanes 7-12 with lanes
1-6), whereas that of p91 was decreased ~16-fold by the drug
(Fig. 5B, compare lanes 7-12 with lanes
1-6). The drug resistance of p67 in relaxation is not due to a
lowered binding affinity of the drug since camptothecin was found to be equally competent in stabilizing cleavage complexes introduced by p67
or p91 on double-stranded DNA fragments (data not shown). Rather, the
N-terminal domain is likely required for camptothecin to block DNA
strand rotation. Truncated versions of human topo I consisting of amino
acids 175-765 or 191-765 have previously been reported to retain
camptothecin sensitivity in relaxation (9, 10, 14). Thus, taken
together the available data indicate that the peptide region spanning
amino acids 191-206 is important for drug sensitivity in relaxation,
which in turn implicates this region in the controlling of strand
rotation.
Temperature Sensitivity of p67--
While optimizing conditions
for assaying the camptothecin effect (Fig. 5), we discovered a striking
difference between p91 and p67 in their sensitivity to low temperatures
(Fig. 6). The two enzyme forms were
roughly equally active at 37 °C. However, at 0 °C the relaxation
rate of p91 dropped ~64-fold relative to that at 37 °C (compare
lanes 22-24 of Fig. 6A with lanes
10-12 of Fig. 6B), whereas the corresponding activity
drop for p67 was much higher, leaving the enzyme almost inert in
relaxation (Fig. 6A, lanes 2-12). At 45 °C,
the relaxation activities of p67 and p91 were comparable (data not
shown), as was the case at 37 °C.
The cold sensitivity of p67 could be due to a conformational change,
leaving the enzyme completely inactive at 0 °C, or any single one of
the catalytic steps could be defective. To investigate these
possibilities, the cleavage and ligation reactions were analyzed
separately at 0 °C. Fig. 7A
shows that the initial rate of cleavage by p67 at 0 °C was decreased
~11-fold relative to p91, whereas there was no significant difference
between the ligation rates of p67 and p91 at 0 °C (Fig.
7B). These experiments show that p67 is capable of
catalyzing transesterification at 0 °C, indicating that the active
site of the enzyme is competent in transesterification chemistry even
at low temperatures. The reduced ability of p67 to mediate strand
cleavage may therefore rather be due to defects in the catalytic steps
before cleavage such as DNA binding and/or in bringing the enzyme-DNA
complex into a proper conformation for cleavage.
Although divergent in amino acid sequence, the N-terminal domain
has been preserved through evolution as a highly basic region in all
cellular type IB topoisomerases (32). In human topo I, a number of
nuclear localization sequences, phosphorylation sites, and protein
interaction sites have been mapped to the N-terminal domain (11,
32-36). These diverse features are suggestive of multiple modes of
topo I regulation in vivo. However, the N-terminal domain
has long been thought to contribute little to the topo I enzyme
activity per se. Here we describe for the first time a
significant modulation of in vitro DNA relaxation mediated
by residues within this domain.
The salient findings from our present work may be briefly summarized as
follows. In the absence of the N-terminal domain, relaxation by human
topo I becomes more distributive. The p67 enzyme is unaffected in
intramolecular ligation but severely depressed in intermolecular
ligation. DNA relaxation by p67 is unimpeded by camptothecin, whereas
p91 is inhibited ~16-fold by the drug. The p67 protein also differs
from p91 in its cold sensitivity in both DNA cleavage and relaxation,
whereas intramolecular strand ligation mediated by p67 is not subject
to this temperature effect. As explained below, all of these
observations can be accommodated by a model according to which residues
within the N-terminal domain participate in the physicochemical steps
of topoisomerization by mediating enzyme-DNA contacts either by binding
DNA directly or by promoting DNA binding of other regions of the enzyme.
The role of N-terminal domain in DNA binding, initially suggested by
the distributive relaxation mode of p67, was further supported by
nitrocellulose binding assays using double-stranded DNA targets, which
demonstrated an inherent DNA binding affinity of this domain. The data
are most consistent with the potential interaction site of DNA
downstream of the cleavage site. Two pieces of evidence argue in favor
of this interpretation: the contrasting effects of the N-terminal
domain in intramolecular versus intermolecular ligation as
well as the ability of p67 to carry out relaxation unimpeded by
camptothecin. A similar effect on camptothecin sensitivity was recently
reported for a form of human topo I lacking the linker domain (amino
acids 636-712) (9). Under the assumption that camptothecin inhibits
DNA relaxation by blocking a putative controlled strand rotation step
of catalysis, the interpretation of this result was that the linker
domain participates in controlling rotation of the cleaved strand
during topoisomerization. Under the same assumption, our results
suggest that the N-terminal domain could also be important for
controlling strand rotation. Thus, based on the available data, we
propose that residues from both the N-terminal and the linker domain
may act together to control this step of catalysis. Two
N-terminal-truncated versions of human topo I encompassing amino acids
175-765 or 191-765 (9, 14) have previously been shown to retain
camptothecin sensitivity in relaxation. In combination with our
results, the region spanning residues 191 through 206 is therefore
highlighted as particularly important for catalysis. Interestingly, the
recently published structure of human topo I including amino acids
203-214 shows the proximal part of the N-terminal domain to be located
close to the cleavage site, supporting that this area of the enzyme could interact with the rotating strand (37). Moreover, circumstantial evidence for our general conclusions on DNA binding is provided by the
presence of 13 lysine residues in the region between residues 175 and
202. In principle, the involvement of the N-terminal domain in
catalysis is well substantiated alone by its apparent ability to
contact DNA downstream of the cleavage site. However, the reduced DNA
affinity of p67 could also, completely or in part, result from the lack
of Trp205, which has recently been shown to interact with a
hinge region within the core domain (37). This interaction is believed
to be important in transition between an open and a closed clamp conformation of topo I upon DNA binding. A possible reduced capacity for this conformational change in the absence of the N-terminal domain
would probably result in an overall decrease in DNA binding affinity
and a reduced ability to coordinate DNA contacts during the individual
steps catalysis.
The initially surprising observation that the p67 enzyme is almost
inert in relaxation and strongly impaired in cleavage at 0 °C while
retaining normal capacity for intramolecular DNA ligation is, in
retrospect, not inconsistent with our interpretations. The cold
sensitivity likely reflects the inability of p67 to induce conformational changes within itself and/or the DNA at low
temperatures. Two non-exclusive explanations for the cold sensitivity
of p67 can be hypothesized. One plausible explanation is that p67 is less flexible than the full-length enzyme due to the lack of
interaction between the hinge region and Trp205, as
discussed above. At low temperatures, the lack of such interaction may
restrain transition of the enzyme from an open to a closed clamp
conformation and thereby reduce the DNA binding affinity. Alternatively
or in combination with the above, the p67 enzyme could be defective in
mediating conformation changes in the DNA substrate, e.g.
bending or partial unwinding of the DNA helix, which may be inherent to
the process of cleavage and topoisomerization. A similar phenomenon has
recently been observed for two cold-sensitive mutants of the DnaA
protein from Escherichia coli (38). These mutants are
defective in initiating DNA replication at 20 °C due to their
inability to unwind the DNA double helix. At higher temperatures the
free energy of the DNA is sufficient to overcome the defect of the mutants.
The in vitro functions of the N-terminal domain of human
topo I suggested by the present study may have important implications for the in vivo activity of the enzyme. It has been shown
that topo I can be regulated via phosphorylation of residues located in
the N-terminal domain (32, 33, 39), which may in fact change the DNA
binding properties of this domain. In addition the relaxation activity
of the enzyme is regulated by a number of proteins: SV40 large
T-antigen, p53, casein kinase II, PSF, nucleolin, non-histone
HMG proteins, and histone H1, all of which interact with and stimulate
topo I activity (34, 40-44). All interaction domains mapped so far
involve the N-terminal domain of topo I (34, 40, 43), and consequently,
the interactions may interfere with the DNA binding properties of this
domain. In support of the N-terminal domain importance for modulating topo I activity in vivo, band depletion experiments using
camptothecin have shown that p67 is strongly impaired in cleavage
in vivo,2
suggesting a role of the N-terminal domain for interaction with DNA
assembled into chromatin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C.
[
-32P]ATP (7000 Ci/mmol) was from ICN. T4
polynucleotide kinase and bovine serum albumin were from New England
Biolabs. pBR322 was from Roche Molecular Biochemicals.
Glutathione-Sepharose columns, Source 15S, thrombin, and pGEX-2TK were
from Amersham Pharmacia Biotech. Nitrocellulose filters were from Whatman.
-D-thiogalactopyranoside for 1 h at 37 °C. Cells
were harvested by centrifugation at 4,000 rpm for 10 min at 4 °C and
resuspended in 10 ml of 1× PBS. Crude cell extract was obtained by
sonication for 1 min on ice. Sonication was repeated 4-5 times
separated by a 1-min incubation on ice, and cell debris was
subsequently eliminated by centrifugation for 5 min at 14,000 rpm. The
supernatant was loaded onto a 2-ml prepacked glutathione-Sepharose
column by gravity flow, and the column was subsequently washed with 20 ml of ice-cold 1× PBS. Before eluting p25 in 500 mM NaCl,
10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, and 10%
glycerol, 15 units of thrombin protease was dissolved in 1.5 ml of 1×
PBS and loaded onto the column, which was then sealed and incubated
overnight at room temperature. Homogeneity of the p25 preparation was
obtained on a 100-µl Source 15S column as described above for p67 and
p100. An equal volume of 87% glycerol was added to the enzyme
preparations before storage at
20 °C. Protein preparations were
analyzed by SDS-polyacrylamide gel electrophoresis. After Coomassie
staining, the intensities of the protein bands were compared with a
dilution series of a bovine serum albumin standard to determine the
protein concentrations.
-mercaptoethanol. Next, the samples were applied to an 8% SDS
Tris-glycine polyacrylamide gel (NOVEX) and were electrophoresed for
2 h at 100 V. Subsequent to electrophoresis, the gel was fixed for
15 min in 10% (v/v) acidic acid and dried.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (16K):
[in a new window]
Fig. 1.
Purification of p67 and p91 forms of topo
I. A, domain organization of p67 and p91. B,
SDS-polyacrylamide gel electrophoresis analysis of 2 µg of purified
p67 and p91 forms of human topo I stained with Coomassie Blue.
View larger version (64K):
[in a new window]
Fig. 2.
Comparison of DNA relaxation activity of p67
and p91. A, relaxation of supercoiled pBR322 by p67 and
p91 in a molar excess of DNA. Lanes 1 and 7, 50 fmol of pBR322. Lanes 2-6, 50 fmol of pBR322 incubated with
17 fmol of p67 at 37 °C for 0.3, 1, 4, 16, and 64 min, respectively.
Lanes 8-12, same as lanes 2-6 using p91 instead
of p67. B, DNA relaxation by p67 and p91 in a molar excess
of enzyme. Lanes 1 and 7, 50 fmol of pBR322.
Lanes 2-6, 50 fmol of pBR322 incubated with 100 fmol of p67
at 37 °C for 10, 20, 40, 80, and 160 s, respectively.
Lanes 8-12, as lanes 2-6 except that p67 was
replaced with p91. SC, negatively supercoiled pBR322.
RL, relaxed pBR322.
View larger version (16K):
[in a new window]
Fig. 3.
DNA binding properties of p25, p67 and
p91. A, salt optimum for DNA relaxation mediated by p67
and p91. 50 fmol of pBR322 was reacted with 5 fmol of either p67 or p91
in the presence of indicated NaCl concentrations for 1 h at
37 °C, and the resulting products were analyzed on a 1% agarose
gel. Subsequent to ethidium bromide staining a digital image of the gel
was acquired. The amount of supercoiled pBR322 remaining at each salt
concentration was quantified and divided by the amount of supercoiled
pBR322 in the control reaction with no enzyme to obtain a measure for
relaxation activity, which is depicted graphically as a function of
salt concentration. B, DNA binding of p25. Binding of p25 to
double-stranded DNA was tested by incubation with 20 fmol of a 100-base
pair fragment of 5'-end labeled duplex DNA (OL100T/OL100B) for 5 min at
room temperature. Enzyme-bound DNA was separated from unbound DNA by
filtration through a nitrocellulose membrane, which retains
specifically protein-DNA complexes. Cytochrome c
(C) was used to demonstrate that a non-DNA-binding protein
with an overall charge similar to that of p25 fails to retain
radiolabeled duplex DNA on the nitrocellulose filter. The resulting
filter of such an experiment is shown in the upper panel.
The amount of radiolabeled DNA retained on the filter was quantified on
a PhosphorImager, normalized to the total amount of radiolabel, and
plotted as a function of the protein concentration (lower
panel). Filled diamonds, p25. Filled
circles, cytochrome c.
View larger version (23K):
[in a new window]
Fig. 4.
DNA cleavage and ligation rates at
37 °C. The cleavage and ligation assays are schematically
depicted in the right panels. The left panels are
graphic representations of the obtained results. A, DNA
cleavage rate for p67 and p91. A radiolabeled suicide DNA substrate was
incubated with the topo I variants for the indicated time periods.
Cleavage products were analyzed by SDS-polyacrylamide gel
electrophoresis, and the percentage of cleaved DNA substrate was
plotted as a function of incubation time. B, rate of
intramolecular DNA ligation by p67 and p91. Active cleavage complexes
containing p67 or p91 attached at an internal position were reacted
with 1 µM dApdG. The reactions were stopped at the
indicated times by the addition of SDS, and the reaction products were
analyzed by denaturing gel electrophoresis. The amount of cleavage
complexes converted to ligation product in each sample was plotted as a
function of time. C, rate of intermolecular DNA ligation by
p67 and p91. Performed as in panel B except that cleavage
complexes containing p67 or p91 attached at a blunt end were reacted
with a 28-mer duplex DNA. Filled triangles, p67.
Filled squares, p91. Asterisk, 5'-radiolabeling.
Filled circles, 5'-phosphorylation. T, topo
I.
View larger version (52K):
[in a new window]
Fig. 5.
Sensitivity of p67 and p91 to
camptothecin. Relaxation of supercoiled pBR322 by p67 and p91
assayed in the absence or presence of 60 µM camptothecin
(CPT). A, sensitivity of p67 to camptothecin.
Samples containing 50 fmol of pBR322 and 100 fmol of p67 were incubated
at 37 °C for indicated periods of time. Lanes 1 and
7, 50 fmol of pBR322. Lanes 2-6, samples stopped
after 0.3, 1, 4, 16, and 64 min of incubation with p67 in the presence
of 5% Me2SO. Lanes 8-12, same as lanes
2-6 but incubated in the presence of 60 µM
camptothecin and 5% Me2SO. B, sensitivity of
p91 to camptothecin. Same as panel A using p91 instead of
p67. SC, negatively supercoiled pBR322. RL,
relaxed pBR322.
View larger version (32K):
[in a new window]
Fig. 6.
Temperature dependence of DNA relaxation
mediated by p67 and p91. Relaxation of supercoiled pBR322 by p67
and p91 at different temperatures. A, DNA relaxation at
0 °C. The reaction mixture was prepared and incubated on ice.
Samples were withdrawn at the indicated times and stopped by the
addition of SDS. Each sample contained 50 fmol of pBR322 and 100 fmol
of either p67 or p91. Lanes 1 and 13, 50 fmol of
pBR322. Lanes 2-12, samples were stopped after 0.2, 0.3, 0.7, 1.3, 2.7, 5.3, 11, 21, 43, 85, and 171 min of incubation with p67.
Lanes 14-24, same as lanes 2-13 but using p91
instead of p67. B, DNA relaxation at 37 °C. The reaction
mixture was prepared and incubated at 37 °C, but otherwise the
treatments were as in panel A. Lanes 2-6,
samples were stopped after 0.2, 0.3, 0.7, 1.3, and 2.7 min of
incubation with p67. Lanes 8-12, same as lanes
2-6 but with p91 replacing p67. SC, negatively
supercoiled pBR322. RL, relaxed pBR322.
View larger version (12K):
[in a new window]
Fig. 7.
DNA cleavage and ligation rates at
0 °C. Cleavage and ligation rates were assayed essentially as
described in the legend for Fig. 4, but with the modification that all
reactions were incubated on ice. A, DNA cleavage rates for
p67 and p91 at 0 °C. The percentage of cleaved DNA substrate is
plotted as a function of incubation time. B, rates of
intramolecular DNA ligation by p67 and p91 at 0 °C. The amount of
cleavage complexes converted to ligation product in each sample is
plotted as a function of time. Filled triangles, p67.
Filled squares, p91. Asterisk, 5'-radiolabeling.
Filled circles, 5'-phosphorylation. T, topo
I.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are grateful to Kirsten Andersen and Inger Bjørndal for skillful technical assistance.
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FOOTNOTES |
---|
Present address: Dept. of Genetics and Development, College of
Physicians and Surgeons, Columbia University, 701 West 168th St., New
York, NY 10032.
§ Present address: Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021.
** To whom correspondence should be addressed. Tel.: 45-89422703; Fax: 45-89422612; E-mail: brk@mbio.aau.dk.
Published, JBC Papers in Press, March 29, 2001, DOI 10.1074/jbc.M010991200
This work was supported by Danish Cancer Society Grants 9710032, 9910012, and 9910013, the Alfred Benzon Foundation, the Danish Research Councils, the Biotechnological Research Program (Biotec III), and the Danish Center for Molecular Gerontology. Support for the Boege laboratory was from Deutsche Forschungsgemeinschaft Grants SFB 172/B12, Bo 910/2-1, and Bo 910/3-1. Support for the Jayaram laboratory was provided by the Robert F. Welch Foundation, The Texas Board for Coordinating Higher Education, and the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2 M. O. Christensen and C. Mielke, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
topo I, topoisomerase I;
p91, human topo I (amino acids 1-765);
p67, human
topo I (amino acids 207-765);
p25, human topo I (amino acids 1-218);
GST, glutathione S-transferase;
PBS, phosphate-buffered
saline;
dApdG, 2'-deoxyadenylyl(3' 5')-2'-deoxyguanosine.
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REFERENCES |
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