(Received for publication, October 3, 1994; and in revised form, January 6, 1995)
From the
The cytotoxic plant alkaloid camptothecin promotes DNA topoisomerase I-linked nicks in DNA by stabilizing a covalently bound enzyme-DNA complex. In the yeast Saccharomyces cerevisiae, substitution of Arg and Ala for the amino acid residues immediately N-terminal to the active site tyrosine in the yeast and human DNA topoisomerase I mutants, top1 vac, results in camptothecin resistance. To examine the mechanism of drug resistance, we assessed the sensitivity of these enzymes to several classes of DNA topoisomerase poisons. Yeast cells expressing the camptothecin-resistant top1 vac mutants were resistant to all of the camptothecin derivatives cytotoxic to wild-type TOP1-expressing cells. This correlated with a significant reduction in drug-induced DNA cleavage in vitro. However, the yeast and human mutant enzymes differed in their responses to the minor groove binding ligand netropsin and to saintopin, a DNA intercalator that targets both DNA topoisomerase I and II. The yeast mutant enzyme demonstrated enhanced sensitivity to the action of saintopin but was resistant to the inhibitory effects of netropsin. In contrast, the human Top1 vac enzyme was resistant to saintopin and indistinguishable from the wild-type enzyme in its response to the netropsin. These results are discussed in terms of enzyme function and the different modes of action of these DNA topoisomerase poisons.
DNA topoisomerases catalyze changes in DNA topology through cycles of transient DNA strand breakage and religation (reviewed in (1, 2, 3, 4) ). During this process, the active site tyrosine in eukaryotic DNA topoisomerase I becomes covalently linked to the 3`-end of a single-stranded nick in the DNA duplex, while DNA topoisomerase II becomes linked to staggered 5`-ends of the cleaved DNA duplex. These enzymes play a critical role in DNA replication and transcription and have been shown to be the targets of an ever increasing number of anti-tumor agents(1, 2, 3, 4, 5) .
Camptothecin is a potent anti-tumor agent that stabilizes the
covalent enzyme-DNA intermediate formed by eukaryotic DNA topoisomerase
I(6, 7) . Drug treatment results in an inhibition of
DNA replication, chromosomal fragmentation, and cell cycle arrest in
G(8, 9) . Since DNA synthesis inhibitors
abolish the cytotoxic activity of
camptothecin(10, 11) , the drug-stabilized enzyme-DNA
adducts are presumably converted into lethal lesions following their
interaction with DNA replication forks(12) . The increased
camptothecin sensitivity of yeast strains defective in the repair of
double-stranded DNA breaks, due to deletion of the RAD52 gene,
further supports this model(13, 14, 15) .
Camptothecin and its derivatives, topotecan (Tpt();
10-hydroxy-9-dimethylaminomethyl-camptothecin), 9-aminocamptothecin
(9-A Cpt), and the pro-drug Cpt-11
(7-ethyl-10-(4-[1-piperidino]-1-piperidino)carbonyloxycamptothecin),
exhibit a broad spectrum of anti-tumor
activity(16, 17, 18, 19, 20) .
The chemical synthesis of camptothecin analogs has identified
structural moieties within the conjugated ring system important for the
anti-tumor activity of the drug, including specific A ring substituents
with increased cytotoxicity(21, 22) . Additional
anti-tumor and anti-fungal agents have been identified that also act as
DNA topoisomerase I poisons (23, 24, 25, 26, 27, 28) .
These include indolocarbazole derivatives (KT6006 and KT6528), minor
groove binding ligands (bulgarein and netropsin), and bisbenzimide dyes
(Hoechst 33258 and Hoechst
33342)(23, 24, 25, 26) . Several
compounds, such as saintopin and intoplicine, trap both DNA
topoisomerase I and II cleavable complexes(27, 28) .
The yeast Saccharomyces cerevisiae has been exploited to
examine the mechanism of action of
camptothecin(13, 14, 15, 29, 30, 31) ,
and specific mutations have been identified in the yeast and human DNA
topoisomerase I genes (ScTOP1 and hTOP1,
respectively) that affect drug
sensitivity(29, 30, 31) . We previously
reported that substitutions in the conserved amino acid residues
preceding the active site tyrosine (Tyr-727) in yeast DNA topoisomerase
I affect the camptothecin sensitivity of these enzymes in vivo and in vitro(30) . In the Sctop1 mutant,
Sctop1NL, exchanging Leu for Asn-726 resulted in
a decrease in enzyme activity and drug sensitivity. Substitution of Arg
for Ile-725 and Ala for Asn-726 in the mutant Sctop1 vac had
no detectable effect on the specific activity of the enzyme, but
dramatically reduced the camptothecin sensitivity of the
enzyme(30, 32) .
In this study, we assessed the
sensitivity of the camptothecin-resistant Top1 vac enzymes to other
topoisomerase poisons, including several camptothecin analogs,
saintopin, Hoechst 22358, and netropsin. Saintopin is a DNA
intercalator that interferes with the catalytic activity of both
eukaryotic DNA topoisomerase I and II(27) . Hoechst 33258 and
netropsin are members of a class of minor groove binding ligands that
preferentially bind AT rich DNA(26) . However, Hoechst 33258,
like DNA intercalators, causes an unwinding of DNA at high
concentrations, while netropsin increases the winding of the DNA
helix(33) . We demonstrate that in comparison to the yeast
wild-type enzyme, the ScTop1 vac enzyme was resistant to all
camptothecin analogs tested, yet exhibited enhanced DNA nicking in the
presence of saintopin and decreased sensitivity to netropsin-mediated
inhibition of DNA relaxation. In contrast, the camptothecin-resistant
hTop1 vac enzyme was resistant to saintopin and indistinguishable from
the wild-type enzyme in its response to netropsin. These results
demonstrate specific alterations in the function of the yeast mutant
enzyme and suggest a model to explain our earlier observations that
expression of Sctop1 vac, but not htop1 vac, produced
sufficient DNA damage in yeast to cause rad52 cell death and
hyper-recombination of the tandemly repeated rRNA genes (31) . ()
S. cerevisiae strains JN2-134 (MATa, rad52::LEU2, trp1, ade2-1, his7, ura3-52, ise1, top1-1, leu2) and JCW1 (MATa, his4-539, lys2-801, ura3-52, top1::HIS4) have been described(15, 30) .
As previously reported(15) , the multicopy yeast vectors YEpGAL1-TOP1 and YEpGAL1-hTOP1 harbor ScTOP1 and hTOP1 cDNA sequences, respectively, under the control of the galactose-inducible GAL1 promoter. The plasmid YEpGAL1-top1 vac was constructed by replacing wild-type ScTOP1 sequences in plasmid YEpGAL1-TOP1 with a 2.9-kilobase pair BamHI-XhoI DNA fragment obtained from plasmid YCpGAL1-top1 vac(30) . The mutated sequences in Sctop1 vac substitute Arg for Ile-725 and Ala for Asn-726. Similar substitutions were engineered N-terminal to the active site tyrosine (Tyr-723) in the human mutant htop1 vac(30) . These mutant sequences were excised from plasmid YCpGAL1-htop1 vac in a SnaB1-BstXI DNA fragment and exchanged for the corresponding wild-type sequences in plasmid YEpGAL1-hTOP1, to yield plasmid YEpGAL1-htop1 vac.
To clone a
preferred DNA topoisomerase I cleavage site, identified in Tetrahymena rRNA gene sequences(34) , the
oligonucleotides 5`-GATCTAAAAAAGACTTAGAAAAATTTTTAAA-3` and
5`-GATCTTTAAAAATTTTTCTAAGTCTTTTTTA-3` were annealed and ligated into
the BamHI site of plasmid pBluescript II KS+
(Stratagene). Plasmid pBlueAK3-1 contained a single insert in the
desired orientation. To facilitate the preparation of single P-end-labeled DNA fragments, the cleavage site in
pBlueAK3-1 was cloned into plasmid pHC624(35) . A BglII site was first introduced into the PvuII site
of pHC624, using commercially available DNA linkers. Following the
sequential addition of BamHI and BglII DNA linkers
into the PvuII sites of plasmid pBlueAK3-1, a 480-base
pair BamHI-BglII DNA fragment containing the strong
DNA cleavage site was excised and inserted into the 1565-base pair BamHI-BglII backbone of plasmid pHC624 to yield
pHCAK3-1.
The cells were subjected to one
freeze/thaw cycle at -80 °C, lysed by vortexing in a Bead
Beater (Biospec), as per the manufacturer's instructions, and the
extracts cleared by centrifugation at 15,000 g for 30
min at 4 °C. Following successive ammonium sulfate fractionations,
the 35-75% ammonium sulfate pellet was resuspended in TEEG
buffer. The conductivity of the sample was adjusted to match that of
TEEG + 0.2 M KCl, and the proteins applied to a 50-ml
phosphocellulose column. The bound proteins were eluted with an
increasing gradient of 0.2-0.8 M KCl in 500 ml of TEEG
buffer and fractions containing DNA topoisomerase I protein, as
determined by DNA relaxation assays, were pooled and adjusted to 0.9 M ammonium sulfate. This sample was applied to a 50-ml
phenyl-Sepharose column pre-equilibrated with TEEG + 0.9 M ammonium sulfate and eluted with a linear 0.9-0 M ammonium sulfate gradient in 500 ml of TEEG. This chromatographic
step yielded homogeneous preparations of ScTop1 and ScTop1 vac
proteins; however, the proteins were further concentrated by a final
phosphocellulose chromatography step. The conductivity of the pooled
phenyl-Sepharose column fractions was adjusted to match that of TEEG
+ 0.2 M KCl. The protein was applied to a 1.0-ml
phosphocellulose column equilibrated with TEEG + 0.2 M KCl, and eluted with 2 ml of TEEG + 0.6 M KCl.
Protein concentrations were determined using the Bio-Rad protein assay,
and individual fractions were adjusted to 50% glycerol and stored at
-80 °C.
A partial purification of human DNA topoisomerase I proteins from 2 liters of galactose-induced cells was achieved by phosphocellulose column chromatography of the 35-75% ammonium sulfate pellet as described previously(30) . The proteins were adjusted to 50% glycerol and stored at -20 °C.
DNA topoisomerase I protein levels and integrity, in all column fractions, were assessed by immunostaining of the proteins fractions following SDS-polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose filters. The yeast and human proteins were visualized following incubation of the blots with specific sera obtained from rabbits or scleroderma patients, respectively, and immunostaining with the appropriate alkaline phosphatase-coupled secondary antibody as described by Promega Corp. The purity of the ScTop1 and ScTop1 vac proteins was assessed by silver staining the proteins resolved in SDS-polyacrylamide gels, as per the manufacturer's directions (Bio-Rad).
To examine the activity of the Cpt-resistant top1 vac mutants in more detail, we wanted to determine the effects of these amino acid substitutions on enzyme sensitivity to other DNA topoisomerase poisons including several camptothecin analogs, minor groove binding ligands such as netropsin (26) and saintopin, a DNA intercalator that stabilizes both DNA topoisomerase I and II cleavable complexes(27) . The Cpt derivatives used in these studies include analogs currently in clinical trials (Tpt, SN-38 (the metabolically active form of Cpt-11), and 9-A Cpt) and several other derivatives containing A ring modifications (10-A Cpt, 11-A Cpt, 12-A Cpt, 9-C Cpt, 9-C,10,11-M Cpt, and 9-A,10,11-M Cpt)(16, 17, 18, 19, 20, 21, 22) .
The drug sensitivity of yeast cells expressing ScTOP1 was assayed in the drug-permeable, rad52 yeast strain JN2-134 (15) . However, as PGAL1 promoted expression of Sctop1 vac is lethal to these strains(30) , it was necessary to reduce the level of Sctop1 vac gene expression to levels tolerated by cells, but still sufficient to render the cells sensitive to Cpt. To achieve this, the pGAL1-TOP1 constructs were cloned into a multi-copy vector. We previously reported that under non-inducing conditions, i.e. growth in glucose, the basal level of ScTOP1 or hTOP1 expression restored the camptothecin sensitivity of JN2-134 cells(15) . In these experiments, JN2-134 cells transformed with the YEpGAL1-TOP1 and YEpGAL1-top1vac constructs were replica plated onto SC ura- glucose plates and overlaid with filter discs impregnated with the drugs indicated. Cell killing was scored by zones of clearing around the filter. As shown in Fig. 1, the growth of cells expressing yeast wild-type DNA topoisomerase I was inhibited by Cpt, 10,11-M Cpt, 10-A Cpt, 9-C Cpt, 9-A,10,11-M Cpt, and 9-C,10,11-M Cpt. However, at this level of ScTOP1 expression, no cytotoxic activity was detected with Tpt, 9-A Cpt, 12-A Cpt, 11-A Cpt, or SN-38 (data not shown). 12-A Cpt has previously been reported to be inactive in stabilizing the covalent enzyme-DNA complex formed by mammalian DNA topoisomerase I (38) and was included in these studies as a negative control. The apparent lack of biological activity of the other compounds in yeast may simply result from a decrease in drug uptake. The same pattern of drug-induced lethality was observed with cells expressing hTOP1. In contrast, cells expressing Sctop1 vac or htop1 vac were unaffected by any of the drugs tested, suggesting that the mutant enzymes are cross-resistant to the biologically active camptothecin analogs (Fig. 1, data not shown). Saintopin was also ineffective in inhibiting the growth of JN2-134 cells expressing yeast or human TOP1 (Fig. 1, data not shown).
Figure 1:
Yeast cells expressing DNA
topoisomerase I mutant, Sctop1 vac, are resistant to Cpt
analog-induced cytotoxicity. As detailed under ``Materials and
Methods,'' replicate plates of JN2-134 yeast cells,
transformed with plasmids YEpGAL1-TOP1 or YEpGAL1-top1 vac, were
treated with filter discs saturated with 10 mM solutions of
the indicated drug or MeSO. A, the order of the
drugs spotted onto the filter discs shown in panelsB and C. B, JN2-134 yeast cells expressing
wild-type ScTOP1. C, JN2-134 yeast
cells expressing Sctop1 vac .
Yeast wild-type DNA topoisomerase I and ScTop1 vac
proteins were purified to homogeneity as described under
``Materials and Methods.'' As previously
reported(30) , the specific activities of the wild-type and
mutant enzyme (2.2 10
units/mg and 2.5
10
units/mg, respectively) were indistinguishable in in
vitro plasmid DNA relaxation assays. ScTop1 (78 units) and ScTop1
vac (76 units) were incubated with a single 3`-end-labeled DNA fragment
in the presence or absence of 50 µM drug and then treated
with SDS to irreversibly trap any covalent enzyme-DNA intermediates.
The DNA products were resolved in 8% polyacrylamide, 7 M urea
gels. As shown in Fig. 2, all of the camptothecin analogs tested
produced a dramatic increase in DNA cleavage when incubated in the
presence of wild-type yeast DNA topoisomerase I, relative to the no
drug controls (compare ScTop1lanes 1, 2,
and 4-6 with lane7 in Fig. 2A and ScTop1lanes1-7 with lane8 in Fig. 2B). Surprisingly, the ``inactive'' 12-A
Cpt (38) was also effective in enhancing yeast DNA
topoisomerase I-mediated DNA nicking (Fig. 2B, lane2). Thus, its lack of cytotoxic activity in yeast cells
might stem from some alteration in drug metabolism, rather than its
inability to form a ternary complex with DNA topoisomerase I and DNA.
In contrast, the ScTop1 vac protein appears to be resistant to all of
the Cpt analogs tested, as samples incubated with equal concentrations
of ScTop1 vac protein consistently exhibited much lower levels of
druginduced DNA cleavage (Fig. 2, A and B).
Figure 2:
The ScTop1 vac enzyme is resistant to Cpt
analog-induced DNA cleavage. As described under ``Materials and
Methods,'' DNA cleavage was assayed in 50-µl reaction volumes
containing 0.5 ng (6,500 cpm) of singly P-end-labeled DNA
and 35 ng of ScTop1 or ScTop1 vac enzyme (78 or 76 units, respectively)
in the presence of 50 mM KCl. In addition, the reactions
contained 50 µM of the following drugs in a final 4%
Me
SO: A, lane 1, Cpt; lane 2,
9-A Cpt; lane 3, saintopin; lane 4, SN-38; lane
5, 10,11-M Cpt; lane 6, Tpt; lane 7, no drug; B, lane 1, Cpt; lane 2, 12-A Cpt; lane
3, 11-A Cpt; lane 4, 10-A Cpt; lane 5, 9-C Cpt; lane 6, 9-A,10,11-M Cpt; lane 7, 9-C, 10,11-M Cpt; lane 8, no drug. Lane C contained DNA substrate
alone. The sites of cleavage (numbered1-12 in A) were mapped by comparison with Maxam-Gilbert
sequencing ladders (data not shown) and placed in rank order in Table 1. The dideoxy sequencing ladder shown was used as a size
marker.
Densitometric quantitation of the major cleavage sites, labeled 1-12, indicated a consistent 20-fold decrease in overall band intensities in the Cpt- and analog-treated ScTop1 vac reactions. For example, while 44% of the DNA was cleaved by ScTop1 in the presence of 50 µM Cpt (Fig. 2A, ScTop1 lane 1), this value decreased to 2.8% in the ScTop1 vac sample (Fig. 2A, ScTop1vaclane1). Similar values (46.5% DNA cleavage by ScTop1 versus 3.6% DNA cleavage by ScTop1 vac) were obtained with 9-A Cpt (Fig. 2A, lanes2). These values include band 2, which corresponds to a high affinity cleavage site for DNA topoisomerase I derived from Tetrahymena rDNA (34) . Although this site is cleaved with greater efficiency by the ScTop1 vac enzyme in the absence of camptothecin, there is significantly less drug-enhanced cleavage at this site by either enzyme than at many other sites. Identical results were obtained with 100 µM drug concentrations (data not shown).
With few
exceptions, the enhancement of specific DNA cleavage sites by the Cpt
analogs was remarkably consistent, with the greatest enhancement
occurring at sites 12, 1, and 4. A comparison of the DNA sequences
surrounding these cleavage sites is depicted in Table 1. The
sites are ordered by decreasing band intensity obtained in the
camptothecin-treated reaction shown in Fig. 2A (ScTop1 lane 1). As reported for mammalian DNA
topoisomerase I(39) , the yeast enzyme preferentially cleaves
at T (position -1), with a marked preference for G at +1 at
sites most enhanced by Cpt. Although the two strongest sites (12 and 1)
had the sequence TGTAAA
, the
significance of this sequence similarity in the absence of any other
consensus is unclear.
The results obtained with saintopin suggest
that the Cpt-resistant ScTop1 vac enzyme is actually more sensitive to
saintopin-enhanced DNA cleavage than wild-type DNA topoisomerase I. A
comparison of band intensities obtained with saintopin-treated ScTop1 (Fig. 2A, lane3) and ScTop1 vac (Fig. 2A, lane3), indicate a
consistent 2-fold increase in the % DNA cleaved (8.7% versus 15.2%, respectively). Similar values were obtained with 100
µM saintopin (data not shown). The DNA sequences cleaved
by either enzyme in the presence of saintopin were largely the same as
those induced by Cpt, although the relative intensities of the cleaved
bands differed. The two strongest sites, 6 and 8, had a TCAT(G/A)G
sequence.
To examine the drug sensitivities of the Cpt-resistant hTop1 vac enzyme, partially purified preparations of equal protein concentration and catalytic activity were incubated in DNA cleavage reactions with 50 µM Cpt, 9-A Cpt, saintopin, SN-38, 10,11-M Cpt, and Tpt (Fig. 3). DNA cleavage by the wild-type human enzyme was enhanced by the addition of Cpt analogs, while hTop1 vac was resistant to Cpt analog-induced cleavage. As with yeast DNA topoisomerase I, the pattern of DNA cleavage obtained with hTop1 in the presence of saintopin was much the same as that observed with Cpt, although the relative intensities of some bands were quite different. However, in contrast to results obtained with various ScTop1 vac preparations ( Fig. 2and data not shown), the hTop1 vac protein was also resistant to saintopin (Fig. 3). Similar results were also obtained following shorter incubation times (data not shown).
Figure 3:
The
hTop1 vac enzyme is resistant to Cpt derivative-induced DNA cleavage.
Partially purified hTop1 and hTop1 vac enzymes, of comparable specific
activity, were assayed in DNA cleavage reactions as described under
``Materials and Methods.'' The reactions contained 0.5 ng
(6,500 cpm) of the singly P-end-labeled and 50 µM of the following drugs in a final 4% Me
SO: lane
1, Cpt; lane 2, 9-A Cpt; lane 3, saintopin; lane 4, SN-38; lane 5, 10,11-M Cpt; lane 6,
Tpt; lane 7, no drug. Lane C contained DNA substrate
alone.
Negatively supercoiled plasmid pBlueAK3-1 DNA was incubated with homogeneous preparations of ScTop1 and ScTop1 vac (Fig. 4) and increasing concentrations of Cpt, saintopin, or netropsin, ranging from 1 to 150 µM. The reaction products were treated with proteinase K, to remove any covalently bound protein and resolved in 1% agarose gels in the presence of 0.1% SDS and 0.5 µg/ml ethidium bromide. Under these conditions, the relaxation of supercoiled plasmid DNA topoisomers and the extent of DNA nicking could easily be measured.
Figure 4:
The
ScTop1 vac enzyme exhibits altered sensitivities to Cpt, saintopin, and
netropsin in DNA nicking assays. As detailed under ``Materials and
Methods,'' drug-induced DNA nicking and inhibition of enzyme
catalytic activity was assayed by incubating purified ScTop1 (78 units
in A and B, and 7.8 units in C) (lanes
a-g) or ScTop1 vac (76 units in A and B, and 7.6 units in C) (lanes
h-m) in reactions containing increasing concentrations of
Cpt (A), saintopin (B), or netropsin (C). The final drug concentrations were: no drug (lanes a and h), 1 µM (lanes b and i), 5 µM (lanes c and j), 10
µM (lanes d and k), 50 µM (lanes e and l), 100 µM (lanes
f and m), and 150 µM (lanes g and n). In A and B, the samples were
adjusted to a final 5% MeSO. Lane C contains
plasmid DNA alone. The reaction products were resolved in a 1% TBE
agarose gel containing 0.1% SDS and 0.5 µg/ml ethidium bromide. The
relative migration of the nicked (N), supercoiled (Sc), and relaxed (R) plasmid DNA topoisomers are
indicated.
As seen in Fig. 4A, the addition of Cpt to reactions containing wild-type yeast DNA topoisomerase I resulted in a concentration-dependent nicking of plasmid pBlueAK3-1 (lanes b-g), beginning at 5 µM and peaking at 50 µM camptothecin. In comparison, no camptothecin-dependent DNA nicking was observed with ScTop1 vac (lanesi-n). The relaxation activity of both yeast enzymes was unaffected at even high camptothecin concentrations as evidenced by the quantitative shift in DNA mobility from that of negatively supercoiled (laneC, labeled Sc) to relaxed plasmid DNA (labeled R). The addition of 0.5 M KCl prior to SDS treatment completely reversed the drug-stabilized DNA cleavage seen at 50 µM Cpt with ScTop1, yet the pattern of enzyme-catalyzed plasmid DNA relaxation remained unchanged (data not shown).
In panel B, saintopin produced a concentration-dependent nicking of the plasmid DNA with both wild-type and ScTop1 vac enzymes, although the effect was more pronounced at lower concentrations with ScTop1 vac than with ScTop1. Densitometric quantitation of the nicked and relaxed DNAs indicate about a 2-fold increase in DNA nicking with ScTop1 vac at 10 and 50 µM drug. This slight but reproducible increment in mutant enzyme-induced DNA nicking is consistent with the DNA cleavage assays shown in Fig. 2A. At higher saintopin concentrations, the catalytic activity of both enzymes was inhibited, though again, ScTop1 vac activity was inhibited at lower drug concentrations. That the shift in plasmid mobility resulted from an inhibition of catalytic activity, rather than a decrease in plasmid DNA linking number due to drug intercalation, was determined by relaxing the DNA prior to drug addition (Fig. 5). Since the plasmid DNA was relaxed at the time of drug addition, saintopin intercalation would unwind the DNA and produce positive supercoiling of the DNA helix. DNA topoisomerase I-catalyzed relaxation of these positive supercoils, following by the removal of the bound saintopin, would result in accumulation of negative supercoils in the plasmid DNA that would be evidenced by a shift in plasmid DNA mobility to that of supercoiled DNA. However, no such shift in plasmid DNA mobility was observed in the ScTop1 or ScTop1 vac reactions (Fig. 5). Thus, the shift in DNA mobility seen in Fig. 4B results from a drug-induced inhibition of catalytic activity.
Figure 5:
Saintopin inhibits DNA topoisomerase I
catalytic activity. Each 20 µl reaction contained 0.3 µg of
plasmid pBlueAK3-1 DNA, 20 mM Tris (pH 7.5), 0.1 mM Na EDTA, 10 mM MgCl
, 50 µg/ml
gelatin, 150 mM KCl, and 78 units of purified ScTop1 protein (a-c) or 76 units of ScTop1 vac protein. Following
incubation at 30 °C for 30 min, the reactions were either
terminated with SDS (lanes a and d), terminated with
SDS and subsequently treated with 150 µM saintopin (lanes b and e), or treated with 150 µM saintopin (lanes c and f). All samples were then
incubated for an additional 30 min at 30 °C. Untreated negatively
supercoiled plasmid DNA is in lane C. The reaction products
were resolved in a 1% agarose gel containing 0.1% SDS and 0.5 µg/ml
ethidium bromide. Nicked (N), supercoiled (Sc), and
relaxed (R) plasmid DNA topoisomers are as
indicated.
The DNA minor groove binders, including netropsin and Hoechst 33258, produced no detectable increase in cleavable complex formation with either the wild-type or ScTop1 vac enzymes (Fig. 4C and data not shown). However, concentrations in excess of 50 µM netropsin severely inhibited the catalytic activity of wild-type yeast DNA topoisomerase I, yet had little effect on ScTop1 vac enzyme activity. Hoechst 33258 inhibition of ScTop1 and ScTop1 vac enzyme activity was detected at the same concentration (50 µM) (data not shown). As with saintopin, similar controls were done to confirm that the shift in plasmid DNA linking number resulted from a drug-mediated inhibition of catalytic activity, rather than relaxation of plasmid DNA in the presence of DNA unwinding or winding agents (data not shown). Thus, ScTop1 vac exhibited enhanced sensitivity to the action of the DNA intercalating agent, saintopin, in inducing DNA cleavage and inhibiting enzyme activity, but was less sensitive to the inhibitory effects of the minor groove binding ligand netropsin.
Partially purified preparations of yeast and human Top1 and Top1 vac proteins were similarly analyzed for any inhibitory effects of saintopin and netropsin. The partially purified yeast enzymes reiterated the results obtained with the homogeneous yeast enzymes (data not shown). In contrast, the concentration dependence of saintopin-mediated hTop1 and hTop1 vac enzyme inhibition were indistinguishable (Fig. 6A). Under reaction conditions optimal for plasmid DNA relaxation activity, the levels of saintopin-induced DNA nicking were too low to detect with partially purified enzyme preparations. Similar results were obtained with netropsin; the catalytic activities of the human wild-type and mutant enzymes were inhibited at the same concentrations (Fig. 6B). Taken together, these data demonstrate distinct differences in the function of yeast and human DNA topoisomerase I mutants. Identical amino acid substitutions in the camptothecin-resistant top1 vac mutants produce dramatic differences, not only in the in vivo activity of these enzymes(30) , but also in their response to other classes of DNA topoisomerase I poisons in vitro.
Figure 6:
Human Top1 and Top1 vac enzymes have
similar responses to netropsin and saintopin in vitro. The
sensitivity of hTop1 and hTop1 vac enzymes to saintopin and netropsin
were determined in nicking assays as described in the legend for Fig. 4and under ``Materials and Methods.'' The
reactions contained hTop1 (lanes a-g) and hTop1 vac (lanes h-n) of equivalent specific activity. Lane C contains DNA alone. The concentrations of saintopin in A and netropsin in B were: no drug (lanes a and h), 1 µM (lanes b and i), 5 µM (lanes c and j), 10
µM (lanes d and k), 50 µM (lanes e and l), 100 µM (lanes
f and m), and 150 µM (lanes g and n). In A, the samples were adjusted to a final 5%
MeSO.
Using the yeast S. cerevisiae, we have begun to examine the effects of specific amino acid substitutions in DNA topoisomerase I on the function and Cpt sensitivity of this highly conserved enzyme (30, 31) . We previously reported that mutation of the SKIN residues preceding the active site tyrosine in yeast DNA topoisomerase I to the SKRA sequence found at the corresponding position in vaccinia virus DNA topoisomerase I (30, 32, 41) reduces the camptothecin sensitivity of the yeast enzyme without affecting the specific activity of the enzyme (30) . Similar changes in human DNA topoisomerase I also produced a catalytically active, camptothecin-resistant enzyme(30) .
Given the current clinical interest in the
anti-tumor activity of camptothecin and several derivatives, including
Cpt-11, Tpt, 9-A Cpt, and 10,11-M
Cpt(18, 19, 20) , and the identification of
additional compounds that specifically target mammalian and fungal DNA
topoisomerases(23, 24, 25, 26, 27, 28) ,
we examined the sensitivity of the camptothecin-resistant Top1 vac
enzymes to a variety of Cpt analogs and representatives of other
classes of DNA topoisomerase I poisons. The growth of drug-permeable, top1, rad52 yeast strains expressing either hTOP1 or ScTOP1, was inhibited by a number of Cpt analogs,
while cells expressing htop1 vac or Sctop1 vac were
resistant. The inability of several Cpt analogs and saintopin to induce
cell death most likely reflects a defect in drug uptake or metabolism
in yeast, since all of these compounds tested in in vitro DNA
cleavage reactions dramatically enhanced the DNA cleavage of yeast and
human DNA topoisomerase I. In contrast to results reported with
mammalian DNA topoisomerase I(38) , the addition of an amino
group at C-12 on the A ring in 12-A Cpt did not inhibit the ability of
this derivative to stabilize the yeast enzyme-DNA complex in
vitro.
The ScTop1 vac and hTop1 vac enzymes were resistant to all of the Cpt derivatives tested. However, the response of these mutant enzymes to other classes of DNA topoisomerase I poisons were quite different. Similar to the results obtained with the camptothecin-resistant htop1 mutant htop1N7225(42) , the hTop1 vac enzyme was cross-resistant to saintopin. In contrast, the ScTop1 vac enzyme exhibited a slight but reproducible increase in sensitivity to saintopin-induced DNA cleavage and inhibition of catalytic activity. Netropsin, on the other hand, was much more effective in inhibiting the catalytic activity of the wild-type yeast, wild-type human, and hTop1 vac enzymes than that of ScTop1 vac. Unlike Cpt, saintopin intercalates into the DNA helix, producing an unwinding of the DNA(27) . Netropsin is a minor groove binding ligand that preferentially binds AT-rich sequences and induces a winding of the DNA helix(33) . Given the opposing effects of these compounds on DNA structure and on the activity of the ScTop1 vac and wild-type enzymes, it is tempting to speculate that drug-mediated changes in DNA structure may induce specific alterations in the interactions of the ScTop1 vac enzyme with its DNA substrate. However, since there is no apparent difference in the sensitivity of these enzymes to Hoechst 33258, a minor groove binding ligand that also appears to have an intercalative mode of DNA binding(25) , and ethidium bromide, another DNA intercalator (data not shown), the differences in enzyme activity and drug sensitivity probably reflect more specific responses to drug-induced local changes in DNA structure.
A similar mechanism may underlie the
differences in the in vivo function of the yeast and human top1 vac mutants. Overexpression of Sctop1 vac and
htop1 vac in RAD52 yeast strains from the PGAL1 promoter on a single copy vector has no obvious deleterious
effects on cell viability. However, similar levels of Sctop1 vac expression, but not htop1 vac, in rad52
strains is sufficient to induce cell lethality(30) . Since
deletion of the RAD52 gene impairs the cells ability to repair
double-stranded DNA breaks, the rad52
dependence of
Sctop1 vac-induced lethality suggests the accumulation of
double-stranded DNA breaks as a result of Sctop1 vac expression. The ability of ScTop1 vac protein to induce DNA damage in vivo is consistent with increased rates of rDNA
recombination scored in
top1, RAD52 cells constitutively
expressing lower levels of Sctop1 vac(30) .
Constitutive expression of htop1 vac, on the other hand,
suppressed the level of rDNA recombination to that seen with wild-type
ScTOP1 and hTOP1(30) .
These
differences may reflect specific changes in the catalytic activity of
the yeast and human mutant enzymes. The altered sensitivity of the
ScTop1 vac enzyme to saintopin and netropsin further suggests that
alterations in the catalytic activity of the yeast mutant enzyme may be
exacerbated by specific changes in local DNA topology or structure.
Such changes could result from the movement of complexes along the DNA,
as proposed by Liu and Wang(43) , or may result from specific
interactions of other complexes with DNA topoisomerase I.
The
description of specific amino acid substitutions in yeast and vaccinia
virus TOP1, which increase the stability of the covalent
enzyme-DNA intermediate(44, 45) , ()provides more direct evidence that specific alterations in
covalent complex formation can cause cell death. However, the low level
increase in DNA nicking observed with ScTop1 vac in the absence of Cpt in vitro is comparable to that seen with human DNA
topoisomerase I. Thus, unless the level of DNA nicking by ScTop1 vac is
selectively elevated in response to specific cellular processes or
local changes in DNA structure, this slight elevation in DNA cleavage
is insufficient to account for rad52 cell death. Experiments
are currently under way to investigate these different possibilities.