Inhibition of Flp Recombinase by the Topoisomerase I-targeting
Drugs, Camptothecin and NSC-314622*
Rikke From
Frøhlich,
Stefan
Gude Hansen,
Michael
Lisby
,
Ian
Grainge§¶,
Ole
Westergaard,
Makkuni
Jayaram§, and
Birgitta Ruth
Knudsen
From the Department of Molecular and Structural Biology,
University of Aarhus, Building 130, C. F. Møllers Allé,
DK-8000 Aarhus C, Denmark and the § Section of
Molecular Genetics and Microbiology, University of Texas, Austin,
Texas 78712
Received for publication, December 20, 2000, and in revised form, January 4, 2001
 |
ABSTRACT |
Recombinases of the
-Int family and type IB
topoisomerases act by introducing transient single strand breaks
in DNA using chemically identical reaction schemes. Recent structural
data have supported the relationship between the two enzyme groups by
revealing considerable similarities in the architecture of their
catalytic pockets. In this study we show that the Int-type recombinase
Flp is inhibited by the two structurally unrelated topoisomerase
I-directed anti-cancer drugs, camptothecin (CPT) and NSC-314622. The
interaction of these drugs with topoisomerase I is very specific with
several single amino acid substitutions conferring drug resistance to
the enzyme. Thus, the observed interaction of CPT and NSC-314622 with
Flp, which is comparable to their interaction with the cleavage complex
formed by topoisomerase I, strongly supports a close mechanistic and
evolutionary relationship between the two enzymes. The results suggest
that Flp and other Int family recombinases may provide model systems
for dissecting the molecular mechanisms of topoisomerase I-directed
anti-cancer therapeutic agents.
 |
INTRODUCTION |
Members of the integrase
(Int)1 family of
site-specific recombinases (
-integrase, P1 Cre recombinase,
Escherichia coli XerC/XerD recombinase, Saccharomyces
cerevisiae Flp, and Zygosaccharomyces rouxii R
recombinases among several others) all carry out conservative site-specific recombination using a basic type IB topoisomerase reaction scheme (1, 2). In the first step of recombination, an active
site tyrosine nucleophile attacks the target phosphodiester bond in DNA
to generate a 3'-phosphotyrosyl linkage and a free 5'-hydroxyl group.
In the second step leading to strand rejoining, the 5'-hydroxyl group
is the nucleophile, and the 3'-phosphotyrosyl bond is its target. The
nucleophilic attack is directed across partner substrates so that
strand ligation occurs in the recombinant configuration, which is
opposed to the typical type IB topoisomerase reaction in which ligation
restores the original phosphodiester bond (3). However, the two
ligation modes are not mutually exclusive, as evident from the fact
that some Int recombinases can relax supercoiled DNA in
vitro under certain conditions while some type IB topoisomerases
can mediate recombination and resolution of Holliday junctions (4-8).
Recent structural data have further consolidated the relationship
between the Int family recombinases and the type IB topoisomerases.
Despite the lack of overall sequence homology between the two enzyme
groups, the tertiary folds within the catalytic domains are strikingly
similar between the Int-type recombinases, including Flp (9) and Cre
(10), and eukaryotic topoisomerase I (topo I) (11-14). Moreover, the
critical catalytic moieties include two nearly identical tetrads,
RHRH/W in the recombinases and RKRH in the topoisomerases, together
with the invariant tyrosine nucleophile (1, 11).
In this study we have further probed the functional relationship
between the Int family recombinase and type IB topoisomerase active sites by investigating the sensitivity of Flp toward
camptothecin (CPT) (reviewed in Refs. 3, 15, and 16) and the newly
synthesized CPT-like agent NSC-314622 (17). CPT and its derivatives are among the most promising anti-cancer drugs available today and have
long been known specifically to target topo I in human cells. Although
NSC-314622 is structurally unrelated to the camptothecins, its mode of
action appears to be similar to that of CPT (17). Both drugs inhibit
the religation step of topo I catalysis, whereas they have no effect on
the cleavage reaction (16-19). For the camptothecins, it is well
established that the drugs do not interact with either topo I or DNA
separately (19). Rather, they form a ternary complex with the cleavage
intermediate, blocking the active site of the covalently bound enzyme
(18, 20). In the present study we demonstrate that the Int type
recombinase, the Flp protein, is inhibited by CPT and NSC-314622. This
is the first report of an enzyme other than eukaryotic topo I being
sensitive toward these drugs. The results demonstrate several
similarities and subtle differences between Flp and topo I in the mode
of inhibition by the two drugs and suggest that the simple members of
the Int family such as Flp and Cre may be exploited to dissect the
molecular action of the topo I-directed anti-cancer drugs.
 |
EXPERIMENTAL PROCEDURES |
Materials--
CPT was purchased from Sigma-Aldrich (C9911), and
NSC-314622 was a kind gift from Dr. Yves Pommier (National Institutes
of Health, Bethesda, MD). Both drugs were dissolved and stored in 100%
Me2SO.
Purification of Flp--
Wild type Flp and Flp(Y343F) were
purified as described by Prasad et al. (21). The
concentrations of the enzymes were estimated according to the procedure
of Lee and Jayaram (22).
Synthetic DNA Substrates--
Oligonucleotides for the
construction of the half-sites were synthesized in an Applied
Biosystems model 380A DNA synthesizer using phosphoramidite chemistry
(23) and purified as described previously (24). The sequence of the
half-site DNA substrate was:
5'-aagcttgcgaagttcctatacttt/3'-acgcttcaaggatatgaaagatct with the Flp binding element written in bold letters. The
sequence of the ligator strand was 5'-tttctagagaataggaacttcggg. The
assembly of substrates containing a 5'-radiolabeled scissile strand and a 5'-cold phosphorylated uncleaved strand was performed as described by
Knudsen et al. (25).
Strand Cleavage and Ligation by Flp--
To investigate the
effect of CPT or NSC-314622 on DNA cleavage mediated by wild type Flp,
~0.5 pmol of enzyme was preincubated for 5 min at room temperature in
a standard Flp reaction buffer (100 mM Tris (pH 7.5), 10%
polyethylene glycol, 80 mM NaCl, 10 mM
mercaptoethanol, and 2 mM EDTA) with or without added drugs as stated in the figure legends. Me2SO was added to
a final concentration of 10% to control reactions to make them
comparable with the samples containing drugs (which were applied as
10× stock solutions dissolved in 100% Me2SO). Subsequent
to preincubation, DNA cleavage was initiated by the addition of 0.02 pmol of the radiolabeled half-site, and incubation was continued for 15 min at 30 °C in a 20-µl reaction volume.
To assay the effect of the drug on wild type Flp-mediated DNA
ligation, active cleavage complexes were generated by incubating 0.5 pmol of the enzyme with 0.02 pmol of the half-site in 20 µl of the
standard reaction buffer for 30 min at 30 °C. The active cleavage
complexes were pretreated with indicated concentrations of drugs for 5 min as described above. Subsequently, ligation was initiated by the
addition of 0.00625 pmol of ligator strand (unless otherwise stated),
and incubation was continued in a 30-µl reaction volume for another
15 min at 30 °C. All reactions were terminated by the addition of
SDS (0.1% final concentration) and were treated with proteinase K (100 µg/reaction for 1 h at 37 °C) prior to ethanol precipitation.
The reaction products were fractionated by electrophoresis in 12%
denaturing (5% bis-acrylamide) polyacrylamide gels, and the reaction
products were visualized by phosphorimaging. Studies of the drug
effects on Flp(Y343F)-mediated reactions were carried out as described
above except that the wild type enzyme was replaced with 0.5 pmol of
Flp(Y343F) plus 30 mM tyramine.
Quantification of Flp Reaction Products--
The amount of
Flp-mediated cleavage and ligation was quantified on a model SF
Molecular Dynamics PhosphorImager by integrating the area under the
curve for each radioactive band using ImageQuant software (Molecular
Dynamics). The cleavage activity was calculated as
C/(C + S), and the ligation activity
was calculated as L/(L + C), where
C is the amount of cleavage product, S is the
amount of substrate, and L is the amount of ligation product.
 |
RESULTS |
Flp-mediated DNA Cleavage and Ligation Are Inhibited by CPT and
NSC-314622--
The sensitivity of Flp toward CPT and NSC-314622 in
the cleavage and ligation steps of catalysis was investigated using a synthetic DNA "half-site" substrate containing a single Flp binding sequence (Fig. 1, B and
D, right panels).
This substrate supports cleavage, but the concomitant ligation
is prevented because of 5'-phosphorylation of the noncleaved DNA strand
and release of the short oligonucleotide containing the 5'-OH end.
However, ligation of the cleavage product to free 5'-OH ends can be
effected by adding excess ligator DNA strands that are able to
form base pair with the noncleaved strand (25).

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Fig. 1.
Inhibition of Flp-mediated cleavage and
ligation by CPT and NSC-314622. A and B show
the drug dose-response relationships of Flp (Wt)-mediated
cleavage and ligation, respectively. The data points are the mean of
three independent experiments. The NSC-314622 concentrations were
0.005, 0.01, 0.05, 0.1, and 0.25 mM. The CPT concentrations
were 0.25, 0.5, 0.75, 1.0, and 2.0 mM. The gel
picture (inset, A) is a representative
example of the result obtained by incubating Flp with 0-0.25
mM NSC-314622 prior to adding the radiolabeled half-site
substrate. S represents the uncleaved substrate, and
C is the cleavage product containing a short 3'-linked
Flp-derived protease-resistant peptide. The bands
surrounding S represent cleavage products that are retarded
in the gel because of partial protease digestion of Flp. The reactions
are schematically represented in B and D,
right panels. The substrate was composed of a
5'-radiolabeled 24-mer scissile strand hybridized to a
5'-phosphorylated 24-mer noncleaved strand. During cleavage, one Flp
monomer activates the scissile phosphodiester bond, while another
monomer donates the active site tyrosine for cleavage. The result is a
dimeric complex with one monomer bound noncovalently to the DNA
substrate and the other covalently linked to the 3'-phosphate end. The
addition of ligator DNA containing a free 5'-OH end induces ligation
with the concomitant release of the covalently bound Flp. C
and D show the drug dose-response relationship of
Flp(Y343F)-mediated cleavage and ligation, respectively. In these
experiments, the cleavage nucleophile was supplied exogenously in the
form of 30 mM tyramine. The right panel
in D is a schematic illustration of the reactions. The only
difference from the wild type reactions is that tyramine substitutes
for the Flp monomer donating the catalytic tyrosine. Reactions
incubated with NSC-314622 and CPT are represented by open
and filled circles, respectively.
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|
The cleavage assays were performed by incubating the enzyme with a
radiolabeled half-site substrate in the presence of increasing concentrations of CPT or NSC-314622 and the products analyzed by
electrophoresis in denaturing polyacrylamide gels followed by
phosphorimaging (an example of a gel picture is shown in Fig. 1A, inset). The extent of cleavage was determined
by densitometric scanning of the reaction products and plotted as a
function of the drug concentration in the reaction mixture. Both drugs
inhibited the cleavage reaction (Fig. 1A), NSC-314622
(open circles) being the more competent inhibitor causing a
50% reduction of cleavage at a concentration of 0.05 mM.
In comparison, 1 mM CPT (filled circles) was
required to reduce Flp-mediated cleavage by 50%. For the ligation
assay, cleavage complexes generated by preincubation of Flp with the
half-site substrate were incubated with a molar excess of the ligator
DNA strands and increasing concentrations of the respective drug. The
dose-response curves for the ligation reaction (Fig. 1B)
demonstrated an inhibition pattern similar to the one observed for
cleavage. An ~50% reduction of activity was observed in the presence
of 0.05 mM NSC-314622 (open circles) or 1 mM CPT (filled circles), respectively. In
comparison, topo I-mediated ligation is reduced by 50% in the presence
of 1 µM CPT (26), whereas the inhibition efficiencies of
NSC-31422 on topo I and Flp appear comparable (17).
The observed inhibition of Flp by CPT and NSC-31466 is, to the best of
our knowledge, the first example of an enzyme, other than the cellular
forms of eukaryotic topo I, being affected by these drugs. This result
is in good accordance with a close mechanistic and structural
relationship between the two enzymes. However, Flp differs from
eukaryotic topo I in one respect. Whereas topo I is drug-resistant
during cleavage (data not shown and Ref. 19), Flp is readily inhibited
in this reaction. The inhibition is not the result of an adverse
effect on Flp-DNA interaction, because gel retardation experiments
showed that noncovalent DNA binding by Flp was unaffected by the
presence of 1 mM CPT or NSC-314622 in the reaction mixture
(data not shown). Thus, camptothecin most likely affects a
precleavage Flp step subsequent to DNA binding or the cleavage
chemistry itself. In this connection it is interesting to note that the
topo I is active as a monomer, whereas it takes a dimer of Flp to be
cleavage competent (27). One Flp monomer (bound adjacent to the
cleavage site) provides the RHRW tetrad to activate the scissile
phosphodiester bond, and the other monomer (bound distally) donates the
tyrosine nucleophile for cleavage (28, 29). The sole function of the
second monomer is to provide the nucleophile in the functional
orientation. This monomer can be replaced by exogenously added tyramine
in a Flp(Y343F)-mediated reaction (30). Cleavage by tyramine generates
a 3'-phosphotyramine linkage, which serves as a substrate for DNA
ligation mediated by a single Flp(Y343F) monomer. Consequently,
Flp(Y343F) acts as a true monomer during both cleavage and ligation,
resembling the monomeric action of eukaryotic topo I, which is
highlighted by the ability of Flp(Y343F) to catalyze the relaxation of
DNA supercoils in the presence of tyramine (8).
To elucidate drug interaction with a single Flp monomer, we
investigated the effect of CPT and NSC-314622 on the
Flp(Y343F)-mediated reactions assisted by tyramine (schematically
represented in Fig. 1D, right panel).
Cleavage was assayed by incubating Flp(Y343F) with the half-site
substrate and 30 mM of tyramine in the presence of
increasing concentrations of the drugs (Fig. 1C). The effect of NSC-314622 (open circles) on Flp(Y343F)-mediated cleavage
was comparable with the effect on the wild type Flp reaction
(compare Fig. 1, panels C and A). On the other
hand, CPT (filled circles) was approximately 2-fold more
efficient at inhibiting Flp(Y343F) than wild type Flp (0.5 mM CPT causing a 50% reduction of tyramine-assisted cleavage). Similar inhibition patterns were observed for the
Flp(Y343F)-mediated ligation reaction (Fig. 1D). The drug
concentrations for 50% reduction in activity were 0.05 and 0.5 mM for NSC-314622 and CPT, respectively.
The inhibition by the drugs of Flp(Y343F)-mediated reactions argues
against their action being directed to the dimeric interface between
Flp monomers. More likely, CPT and NSC-314622 interfere with Flp
catalysis by interacting with the pro-active site (the active site that
has to acquire the tyrosine nucleophile in trans to
become cleavage competent) harbored by the Flp monomer. This mode of
action would agree with what has previously been established for
eukaryotic topo I (31). The difference between the two enzymes in drug
sensitivity during cleavage may be explained by the catalytic pocket of
Flp having a more open conformation than that of topo I. Such a
conformation would be required to permit the entrance of the
tyrosine nucleophile in trans from a second Flp monomer and
could facilitate interaction with the active portion of the drug
molecules. This notion is supported by the crystal structure of Flp
showing that within a single monomer the active site tyrosine points
away from the RHRW tetrad, leaving the catalytic pocket in an open
conformation (9).
CPT and NSC-314622 Inhibit Flp Transesterification in a
Competitive Manner--
In an attempt to verify the suspected drug
interaction with the catalytic pocket of Flp, we tested whether
inhibition by CPT and NSC-314622 was competitive with the cleavage and
ligation nucleophiles. For this purpose, DNA cleavage and ligation were assayed in the presence of varying concentrations of substrates (the
nucleophile for transesterification) and drugs relative to each other.
To vary the concentration of the cleavage nucleophile without changing
enzyme concentration, we utilized tyramine-assisted cleavage mediated
by Flp(Y343F) of a half-site substrate. A range of tyramine
concentrations (from 5 to 70 mM) was employed in the presence of different concentrations of either CPT (from 0 to 1 mM) or NSC-314622 (from 0 to 0.1 mM). The
results are depicted as Lineweaver-Burk plots in Fig.
2, A and B. The
mode of inhibition of ligation was investigated in a similar manner
using the strand joining reaction in a cleaved half-site substrate as
described above. Because the natural nucleophile during ligation
is a 5'-OH end of DNA, it was possible to assay this step using the
wild type enzyme. The concentrations of the ligator strand ranged from 0.005 to 1 pmol in the presence of 0-1 mM CPT or 0-0.1
mM NSC-314622. The Lineweaver-Burk plots of the data are
shown in Fig. 2, C and D. The intercepts on the
ordinates of the plots for both cleavage (Fig. 2, A and
B) and ligation (Fig. 2, C and D) were
nearly the same regardless of the concentrations of either CPT or
NSC-314622. Thus, the inhibitions of cleavage and ligation were
competitive in nature and could be overcome by a sufficiently high
concentration of either tyramine or the ligator strand, respectively.
We therefore believe that the sites of occupancy of the drugs and the
incoming nucleophile (tyramine or the 5'-OH DNA end) are the same,
partially overlapping or at least in close enough proximity to elicit
mutual competition. Such a drug interaction mode with Flp is consistent with previously published results demonstrating that CPT inhibits topo
I-mediated ligation in a competitive manner (31). Moreover, the
similarity in the inhibition of Flp-mediated cleavage and ligation
suggests that the two reactions are catalyzed by similar active site
conformations. In both instances, the pro-active site of the Flp
monomer orients the phosphodiester (the DNA phosphodiester or the
phosphotyrosine bond formed by strand cleavage) so that it can be
targeted by the attacking nucleophile supplied in trans (the
active site tyrosine or the 5'-OH from the cleaved strand).

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Fig. 2.
Inhibition by CPT or NSC-314622 of
Flp-mediated cleavage and ligation is competitive.
Flp(Y343F)-mediated cleavage by tyramine and Flp
(Wt)-mediated ligation was assayed in the presence of
varying concentrations of nucleophile (tyramine or ligator DNA) and
drugs (CPT or NSC-314622) relative to each other. The data are
represented as Lineweaver-Burk plots. Results of tyramine (5-70
mM)-assisted cleavage by Flp(Y343F) are shown in
A and B. Reactions shown in A and
B represent the effects of CPT and NSC-314622, respectively,
at the indicated concentrations. The inhibitions of Flp-mediated
ligation reactions by CPT and NSC-314622 are displayed in C
and D, respectively.
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Inhibition of Flp by CPT and NSC-314622 Is Reversible--
It is
well documented that CPT interaction with topo I is transient and that
the effect of high drug concentrations can be reversed by dilution
(19). To further compare Flp and topo I with respect to drug
interaction, we investigated whether inhibition of the Flp recombinase
is also reversible upon drug dilution. Active cleavage complexes
(obtained as described earlier) were split into three samples of 60 µl each and incubated for 5 min without added drug or in the presence
of 2.0 mM CPT or 0.25 mM NSC-314622. At these
drug concentrations DNA ligation was abolished (see Fig. 1). Each
sample was then split into halves. To the first half of each
sample, the ligator strand was added directly to a final
concentration of 200 nM, and incubation was continued for
60 min. The other halves were diluted to a final volume of 1200 µl
(drug concentrations diluted to 0.05 mM CPT or 0.006 mM NSC-314622) and then incubated with 200 nM
ligator strand. The ligation results are depicted by a bar chart (Fig.
3). The almost complete inhibition by the
drugs in the undiluted samples (Undiluted, compare
white and gray bars with
black bar) is in contrast with the nearly
complete recovery of ligation activities upon drug dilution
(Diluted, compare white and gray
bars with black bar).

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Fig. 3.
Inhibition of Flp by CPT and NSC-314622 is
reversible. Active Flp cleavage complexes were preincubated
in the absence of drug or in the presence of 2 mM CPT or
0.25 mM NSC-314622, respectively. The ligation activity of
each preincubation mixture with or without 20× dilution was assayed in
the presence of 200 nM ligator DNA strand (as
described under "Results"). The bars on the
left and right represent the undiluted and
diluted reaction sets, respectively. Within each set, the
bars from left to right indicate
controls without any drug ( ), reactions containing CPT ( ), and
reactions containing NSC-314622 ( ), respectively.
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 |
DISCUSSION AND CONCLUSIONS |
In the present study we have demonstrated that CPT and NSC-314622
inhibit Flp recombinase by blocking the active site from the incoming
nucleophile for strand cleavage or ligation. The fact that two
structurally distinct topo I-directed drugs also inhibit Flp
recombinase argues that their interaction with Flp is unlikely to be
mere coincidence. Rather, the active site of Flp may share sufficient
structural similarities to that of eukaryotic topo I to be able to bind
the two drugs specifically, although Flp differs from topo I regarding
drug affinity. Flp is less sensitive than topo I toward CPT (26), but
the effect of NSC-314622 on the two enzymes appears comparable (17).
Drug interaction with topo I is very specific as revealed by several
single amino acid mutations conferring drug resistance to the enzyme
(32-36) and by topo I from vaccinia virus being drug-resistant because
of a single amino acid difference compared with the cellular forms of
topo I (37, 38). The extent of structural similarities and differences
between Flp and human topo I seen in recently published crystal
structures is comparable with those between Flp and other members of
the
-Int recombinase family (9, 12, 13). Our data are consistent
with the notion that at least some recombinases of the Int family,
including Flp, may have evolved in parallel with the type IB
topoisomerases from a common, and possibly drug-sensitive, ancestral topoisomerase.
Studies with eukaryotic topo I have shown that CPT interacts with the
enzyme only after formation of the covalent cleavage intermediate (19)
and that the inhibitory effect is enhanced by a G-positioned 3' to the
cleavage site (39-41). Moreover, affinity labeling experiments have
indicated that the drug interacts with both the enzyme and the DNA
within the cleavage complexes (18, 20). It has therefore been proposed
that the drug forms a ternary complex with the cleavage intermediate,
with the base 3' to the cleavage site playing a direct role in drug
binding (18, 20). Our results with Flp can be reconciled with the topo
I data if drug accessibility is facilitated by an open post-cleavage
conformation of the topo I active site. This transition may be required
for the entry of the DNA nucleophile (5'-OH) for the strand joining reaction. Because of the trans cleavage mechanism of Flp,
the pro-active site of the Flp monomer has an inherent open
conformation (9), allowing drug binding both in the pre- and
post-cleavage states of the enzyme. Thus, unlike topo I, Flp is drug
sensitive in both the strand cleavage and ligation steps. Although the
conformation of the catalytic pocket of the enzyme may determine the
initial interaction with the drug, this interaction could be further
reinforced by additional drug-DNA interactions. Consistent with such a
mechanism, the presence of DNA downstream to the cleavage site in topo
I-DNA complexes has been found not to be a prerequisite for, even
though it stimulates, the action of camptothecins (26).
Our findings suggest that Flp and other related recombinases that
utilize the type IB topoisomerase mechanism can be exploited successfully to study the mechanism of action of an important class of
anti-tumor drugs.
 |
ACKNOWLEDGEMENT |
We are grateful to Dr. Yves Pommier (National
Institutes of Health, Bethesda, MD) for kindly providing the NSC-314622
for these studies and to Kirsten Andersen for skillful technical assistance.
 |
FOOTNOTES |
*
This work was supported by the 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
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.
Present address: Dept. of Genetics and Development, College of
Physicians and Surgeons, Columbia University, 701 West 168th St., New
York, NY 10032.
¶
Present address: Clare Hall Laboratories, Blanche Lane, South
Mimms, Potters Bar, Hertfordshire EN6 3LD, United Kingdom.
To whom correspondence should be addressed. Tel.:
+45-89422703; Fax: +45-89422612; E-mail: brk@mbio.aau.dk.
Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.C000901200
 |
ABBREVIATIONS |
The abbreviations used are:
Int, integrase;
CPT, camptothecin;
NSC-314622, 5,6-dihydro-5,11-diketo-2,3-dimethoxy-6-methyl-8,9- methyllenedioxy-11H-indenol(1,2-c)isoquinoline;
topo I, topoisomerase I.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.