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J. Biol. Chem., Vol. 276, Issue 28, 26708-26714, July 13, 2001
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,From the Molecular Pharmacology Department, St. Jude Children's Research Hospital, Memphis, Tennessee 38105
Received for publication, March 26, 2001, and in revised form, May 10, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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DNA topoisomerases play essential roles in many
DNA metabolic processes. It has been suggested that topoisomerases play
an essential role in DNA repair. Topoisomerases can introduce DNA damage upon exposure to drugs that stabilize the covalent
protein-DNA intermediate of the topoisomerase reaction. Lesions
in DNA are also able to trap topoisomerase-DNA intermediates,
suggesting that topoisomerases have the potential to either assist in
DNA repair by locating sites of damage or exacerbating DNA damage by
generation of additional damage at the site of a lesion. We have shown
that overexpression of yeast topoisomerase I (TOP1) conferred hypersensitivity to methyl methanesulfonate and other DNA-damaging agents, whereas expression of a catalytically inactive enzyme did not. Overexpression of topoisomerase II did not change the
sensitivity of cells to these DNA-damaging agents. Yeast cells lacking
TOP1 were not more resistant to DNA damage than cells expressing wild type levels of the enzyme. Yeast topoisomerase I
covalent complexes can be trapped efficiently on UV-damaged DNA. We
suggest that TOP1 does not participate in the repair of DNA
damage in yeast cells. However, the enzyme has the potential of
exacerbating DNA damage by forming covalent DNA-protein complexes at
sites of DNA damage.
DNA topoisomerases catalyze the interconversion of topological
isomers of DNA (1). Topological changes catalyzed by these enzymes are
required for a wide variety of cellular processes including
transcription, replication, and chromosome segregation (2-4). The
importance of topoisomerases in DNA metabolism has frequently led to
the suggestion that topoisomerases might play important or essential
roles in DNA repair and DNA damage tolerance. However, there has been
little direct evidence that topoisomerases play a direct role in the
repair of DNA damage in eukaryotic cells (reviewed in Ref. 5).
DNA topoisomerases are the targets of a large number of anti-cancer and
anti-bacterial agents (6, 7). These agents stabilize a covalent
intermediate where the enzyme is covalently bound to DNA through a
phosphotyrosine linkage and, therefore, convert the enzyme into a DNA
adduct with protein bound to the site of DNA strand breaks (8).
Although the covalent intermediate is reversible, DNA metabolic
processes such as replication can convert the intermediate into
irreversible DNA damage. Extensive evidence has demonstrated that the
DNA damage, rather than inhibition of enzyme activity, is responsible
for cytotoxicity (9, 10). Hence these agents have been termed
topoisomerase poisons. Thus, topoisomerases clearly have the potential
of inflicting cytotoxic DNA damage under appropriate circumstances.
Recent experiments demonstrate that alterations in DNA structure are
able to trap topoisomerases on DNA. Topoisomerase I can be trapped by
strand discontinuities such as nicks or gaps (11) or by mismatched
bases (12). UV damage to DNA also efficiently traps eukaryotic
Top1 on DNA (13). Other types of DNA damage such as abasic sites
and ethenoadenine adducts also stabilize Top1 covalent complexes
(14-16). Interestingly, there are two different mechanisms that
can lead to topoisomerase I covalent complexes on DNA. UV damage,
abasic sites, and mismatches all lead to a covalent complex that is not
readily reversible. Other DNA lesions such as oxidized bases or
benzo[a]pyrene adducts increase the rate of cleavage of
the enzyme at or near the lesion but do not prevent re-ligation (17).
This latter mechanism has also been observed for topoisomerase II at
abasic sites (18, 19). Other types of DNA damage such as UV damage
inhibit topoisomerase II enzymatic activity but do not lead to
increased topoisomerase II covalent complexes (20).
If DNA damage is able to trap topoisomerases on DNA in the same way as
topoisomerase poisons, then topoisomerases may influence cell survival
after DNA damage and may also influence the consequences of DNA
lesions. Experiments described here test the hypothesis that the level
of topoisomerases affect cell killing after DNA damage. We have taken
advantage of the fact that yeast cells can tolerate different levels of
both topoisomerase I and topoisomerase II. We have found that
topoisomerase I overexpression greatly sensitizes yeast cells to DNA
damage due to simple alkylating agents, UV light, or ionizing
radiation, but overexpression of topoisomerase II does not affect yeast
cell survival after exposure to these agents. These results indicate
that topoisomerases can be important survival factors after DNA damage
but that the enzymes do not participate directly in repair.
Yeast Strains and Plasmids--
The yeast strains used in this
study are derivatives of CH335 (21). CH335leu was constructed by
converting CH335 to leu2 Determination of Sensitivity to DNA-damaging
Agents--
Sensitivity of yeast cells to
MMS1 was performed as
described previously (10) with the following modifications. Cells were pre-grown in synthetic complete medium without uracil, with galactose as a carbon source (SC-ura/GAL). After overnight growth, cells were
diluted to 2 × 106 cells/ml in fresh SC-ura/GAL, and
then appropriate concentrations MMS were added. Cells were incubated
for various times at 30 °C with shaking, then aliquots were removed,
and diluted samples were plated to synthetic complete agar lacking
uracil, with glucose as carbon source (SC-ura/Glu). Survival is
expressed relative to the number of viable colonies at the time of MMS
addition. For the comparison of the sensitivity of
top1
Sensitivity to ionizing radiation was determined by pre-growing cells
in SC-ura/GAL as described above to a titer of about 107
cells/ml. Cells were washed in water and resuspended in water at a
concentration of about 107 cells/ml. Cells were exposed to
ionizing radiation using a 137Cs irradiator at a flux of
0.77 krad/min. Dilutions were then plated to SC-ura/Glu, incubated for
3 days at 30 °C, and then counted to determine the surviving fraction.
Sensitivity to UV light was determined by pre-growing cells in
SC-ura/GAL as described above to a titer of about 107
cells/ml. Cells were diluted and plated to SC-ura/Glu. The plates were
immediately exposed to UV light using a Stratalinker (Stratagene) and
subsequently protected from visible light to prevent photoreactivation. Plates were incubated for 3 days at 30 °C, then plates with an appropriate number of colonies were counted, and surviving fractions compared with unirradiated plates were determined.
Northern Analysis of RAD54 Expression--
Yeast cells were
exposed to MMS under the same conditions as described for survival
determinations. Total yeast RNA was isolated using the acid phenol
method (27). Total RNA (20 µg/sample) was separated by
electrophoresis in 1% agarose gels containing 2.2 M
formaldehyde. The RNA was transferred onto a nylon membrane by
capillary transfer and UV cross-linked to the membrane. Hybridization was overnight at 60 °C in 0.25 M
NaH2PO4, pH 7.4, 1 mM EDTA, and 7%
SDS with a [32P]dCTP-labeled probe.
32P-Labeled probes were prepared by random priming using a
2-kilobase BamHI fragment of the yeast RAD54
gene. After hybridization, the membrane was washed 3 times at 65 °C
for 15 min in 0.1× SSC (1× SSC = 0.15 M NaCl and
0.015 M sodium citrate), 0.1% SDS and then exposed
to Kodak Bio-Max film at Purification of Yeast Topoisomerase I--
Yeast topoisomerase I
was purified using the procedure described by Bjornsti and
co-workers (28) using the plasmid pGALyTOP1 transformed into strain
JEL1t1 Assessment of Topoisomerase I-DNA Covalent Complexes with
UV-damaged DNA Using K+/SDS Precipitation--
Formation
of covalent complexes by yeast topoisomerase I was assessed using a
modified K+/SDS assay. Negatively supercoiled pHOT1 DNA
(TopoGen, Inc.) was used as a substrate for this assay. pHOT carries
the strong topoisomerase I binding site identified by Westergaard and
co-workers (29). The plasmid DNA was diluted to
A260 = 0.05 in 50 mM Tris-Cl,
pH 8.5. The DNA was UV-irradiated in a 100-mm2 tissue
culture dish on ice using a Stratalinker 2400 (Stratagene, 254 nm) to a
final UV dose of 1000 J/m2. The DNA was then treated with
an ATP-dependent exonuclease and purified using a
large-construct kit protocol (Qiagen) to remove nicked DNA. The DNA was
then digested with EcoRI and labeled using the Klenow
fragment of DNA polymerase I and [ Assessment of Topoisomerase I/DNA Covalent Complexes with
UV-damaged DNA Using an Electrophoretic Mobility Shift
Assay--
Negatively supercoiled pHOT1 DNA was linearized with
EcoRI, diluted in water, and UV irradiated as described
above. After UV treatment, the DNA was ethanol-precipitated. The
topoisomerase I mobility shift assay conditions were as follows. Each
reaction mixture (40 µl total volume) contained 250 ng of
EcoRI-linearized pHOT1 DNA, 10 mM Tris-Cl, pH
7.5, 2 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, pH 8.0, 15 µg/ml acetylated bovine serum
albumin, the indicated units of yTOP1 protein, and as indicated, 20 µg/ml camptothecin. The salt concentration was then adjusted to 70 mM with KCl for each reaction. The mixtures were incubated
for 10 min at 30 °C. Reactions that were to be treated with
proteinase K were stopped with 0.5 µl of 20% SDS; other sample
reactions were stopped with 2 µl of 20% SDS. Proteinase K was added
to a final concentration of 250 µg/ml in samples as indicated, and
samples containing proteinase K were incubated overnight at 50 °C.
All samples received 5 µl of a loading buffer without EDTA (60%
sucrose and 0.67% Orange G) and were analyzed on a 1% agarose gel run
for 400 V-h in Tris acetate EDTA (TAE) buffer. After electrophoresis,
the gel was stained with ethidium bromide, and the band corresponding
to free DNA was excised (to minimize interference with the
hybridization). The DNA contained in the upper portion of the gel was
transferred to a nylon membrane (Hybond-N, Amersham Pharmacia Biotech).
The blot was hybridized overnight with a radiolabeled 500-base pair EcoRI-SspI pHOT1 DNA fragment. The blot was
washed 3 times for 20 min in 0.1× SSC, 0.1% SDS at 65 °C and
exposed to film. Gel shift DNA band intensities were quantitated using
a STORM 860 system and an image quantification program (ImageQuant;
Molecular Dynamics).
Topoisomerase I Overexpression Increases Sensitivity to
DNA-damaging Agents--
The effect of topoisomerase I in the presence
of DNA damage was first examined by overexpressing this enzyme in
Saccharomyces cerevisiae. A plasmid carrying the yeast
topoisomerase I gene under the control of the inducible GAL1
promoter (24) was transformed into yeast strain CH335. The control
cells for these experiments were CH335 cells transformed with the
centromeric vector yCP50. Actively growing cells were exposed to MMS in
SC-ura/GAL. MMS concentrations were selected that reduced the viability
of wild type cells (cells not overexpressing Top1p) to about 10-100%
after 1-3-h exposures. After exposure to MMS, cells were diluted and plated to SC-ura/Glu. The results obtained with MMS are shown in Fig.
1. At different concentrations of MMS,
cell survival was significantly lower in cells overexpressing Top1p
than in cells carrying yCP50. A similar experiment was performed using
yeast cells overexpressing human topoisomerase I from the yeast
GAL1 promoter. Yeast cells overexpressing hTOP1 were also
more sensitive to the killing effects of MMS than cells that did not
overexpress TOP1 (data not shown). Thus, the results
obtained appear to be general for type 1B topoisomerases and are not
due to peculiar properties of yeast topoisomerase I.
The enhanced sensitivity to DNA-damaging agents was not confined
to simple alkylating agents. Similar results were obtained with other
types of DNA damage. Yeast cells overexpressing Top1p exposed to either
UV light or ionizing radiation exhibited significantly reduced survival
when compared with cells carrying the control plasmid yCP50 (Fig.
2, panels A and
B).
Interestingly, a somewhat different pattern of sensitivity was seen
with cells that express E. coli topoisomerase I, a type IA
enzyme. Cells expressing E. coli topA from the yeast
GAL1 promoter had slightly greater sensitivity to MMS than
control cells (Fig. 3). The difference in
sensitivity at 0.04% MMS was statistically significant, whereas the
difference at 0.08% MMS was not statistically significant. Although
expression of a type IA enzyme causes a slight increase in sensitivity
to DNA-damaging agents, the effect is considerably smaller than seen
when eukaryotic type 1B enzymes are overexpressed.
Sensitivity to DNA-damaging Agents Is Unaffected in Cells
Expressing Elevated Levels of Inactive Topoisomerase I--
We next
examined whether the sensitization of cells by TOP1
overexpression requires that the protein be active and able to cleave
DNA. We introduced a plasmid carrying a mutant of TOP1 where
the active site tyrosine (Tyr-727 (30)) was mutated to phenylalanine.
The mutant TOP1 gene was also under the control of the
GAL1 promoter (31). Unlike the results obtained with the
active TOP1 gene, expression of the Y727F mutant did not
sensitize cells to MMS (Fig. 4). It is
also noteworthy that the Y727F mutant had a similar effect on growth in
the absence of MMS as overexpression of the active TOP1 gene
(compare Fig. 1 and Fig. 4). This result shows that the observed
sensitization by TOP1 overexpression is not due to a
reduction in growth rate or decreased plating efficiency when
TOP1 is overexpressed.
Sensitivity to DNA-damaging Agents Is Unaffected by the
Overexpression of Topoisomerase II--
When topoisomerase II is
transcribed at the high levels expressed from the GAL1
promoter, cells are unable to successfully undergo cell division. To
examine the effects of topoisomerase II overexpression in the presence
of DNA-damaging agents, a plasmid containing the topoisomerase II gene
in front of the constitutive promoter DED1 was transformed
into yeast (10). Cells were then treated with MMS, and survival was
measured. The results, shown in Fig. 5,
indicate that expression of the yeast TOP2 gene from the
DED1 promoter does not lead to an increase in sensitivity to
MMS. Similar results were obtained with UV and ionizing radiation (data
not shown). These results suggest that the increase in sensitivity to
DNA damage that is observed with topoisomerase overexpression is
limited to type I topoisomerases.
Induction of the Recombinational Repair Gene RAD54 Is Not Affected
by Overexpression of Topoisomerase I--
Since topoisomerase I has
been implicated in transcription (32, 33), a plausible model for the
effects of topoisomerase I on DNA damage response is an alteration in
the expression levels of genes required for responding to DNA damage. A
set of genes has been identified in yeast whose transcription is
increased after DNA damage (34, 35). We examined the transcription of one such gene, RAD54, which is inducible by MMS. CH335 cells
carrying pGALTOP1 were treated with MMS in either glucose or galactose, and at various times after MMS addition, aliquots were removed for RNA
isolation. The RNA was electrophoresed, and after transfer to a nylon
membrane, probed with full-length RAD54 DNA. The results are
shown in Fig. 6. In glucose in the
absence of MMS, RAD54 mRNA is barely detectable. After a
1- or 3-h exposure to 0.05% MMS, there is a clear induction of the
RAD54 message. Similarly, RAD54 message is barely
detectable in galactose grown cells in the absence of MMS. This
suggests that overexpression of TOP1 from the
GAL1 promoter by itself is insufficient to elicit the
induction of RAD54. Upon the addition of MMS,
RAD54 message is induced to an extent similar to that seen
with glucose-grown cells. Similar results were also obtained with the
gene for a subunit of the single-stranded DNA-binding protein
RPA (data not shown). These results indicate that overexpression
of TOP1 does not increase the sensitivity of cells to
DNA-damaging agents by changing gene expression.
top1 Trapping of Yeast Topoisomerase I by DNA Damage--
Previous
reports discussed in the Introduction have shown that mammalian
topoisomerase I can be trapped on DNA carrying various types of DNA
damage. We next wanted to confirm that yeast topoisomerase I could also
be trapped by DNA damage. We first carried out a simple assay to test
whether there was preferential nicking of damaged DNA by DNA
topoisomerase I. Purified yeast topoisomerase I was incubated with
end-labeled DNA that was either unirradiated or UV-irradiated with 1000 J/m2. After incubation at 30 °C, the reaction was
stopped with SDS, protein-DNA complexes were precipitated by the
addition of excess KCl, and the samples were washed as described under
"Experimental Procedures." Topoisomerase I that was trapped as a
covalent complex caused the bound DNA to precipitate, whereas
free DNA remained in the supernatant. The results of this experiment
are shown in Fig. 8. The addition of
camptothecin to unirradiated DNA samples increased the level of
topoisomerase I-DNA complexes that can be precipitated in the presence
of potassium and SDS. Similarly, using DNA irradiated with 1000 J/m2 UV light in the absence of camptothecin also
efficiently trapped topoisomerase I. In fact, the UV-damaged DNA
trapped topoisomerase I as effectively as camptothecin. We also
observed that the addition of camptothecin to samples containing
irradiated DNA further increased trapping by topoisomerase I over the
level seen with UV-irradiated DNA in the absence of camptothecin.
To be able to examine cleavage under a wider range of conditions or
with DNA substrates containing different types of DNA damage, we
adapted the concept of changing the electrophoretic mobility of DNA
upon protein binding to measure protein-DNA covalent complexes. In a
standard electrophoretic mobility shift assay, protein and DNA are
co-incubated, and then the reaction mixture is analyzed by gel
electrophoresis. Detection of protein-DNA complexes when the
interactions are noncovalent requires that the interaction remain
stable under the electrophoresis conditions. Since we were interested
in quantitating covalent protein-DNA interactions, it seemed likely
that electrophoretic mobility shift would be able to readily detect
topoisomerase I-DNA covalent complexes. To assess this, we examined the
ability of topoisomerase I to reduce the mobility of linear DNA that
was either unirradiated or irradiated with different UV doses. Fig.
9A shows the result of the
electrophoresis. As can be readily seen, a weak shifted band can be
observed in samples containing 6 units of topoisomerase I and
unirradiated DNA. The addition of camptothecin greatly increased the
intensity of the shifted band. If instead of camptothecin, UV-irradiated DNA was used, a significant increase in intensity of the
shifted band was also observed. The intensities of the bands with
unirradiated DNA, DNA irradiated with 1000 J/m2, or 2000 J/m2 UV is shown in Fig. 9B. The intensity of
the band is approximately linear with respect to added topoisomerase I
over the range examined for all three DNA samples. The slope of the
calculated linear regression is 2.8-fold higher for DNA irradiated with
1000 J/m2 than for unirradiated DNA and 5.3-fold higher for
DNA irradiated with 2000 J/m2. Since the reactions were
treated with SDS before electrophoresis, the interactions between
topoisomerase I and damaged DNA that we detect must be covalent rather
than noncovalent. To verify that the shifted bands represent
protein-DNA complexes, we also treated one set of samples containing
the highest amount of topoisomerase I with proteinase K before
electrophoresis. Treatment with proteinase K resulted in complete loss
of the shifted band whether complexes were trapped with camptothecin or
UV damage. The results of Figs. 8 and 9 taken together demonstrate that
UV-damaged DNA can efficiently trap topoisomerase I covalent
complexes.
Anti-cancer drugs such as camptothecin are able to trap a covalent
intermediate of the topoisomerase I reaction, and trapping of this
intermediate can interfere with DNA metabolism. It is well established
that the cytotoxicity of camptothecin depends on its ability to
stabilize topoisomerase I-cleavable complexes and that the degree of
cytotoxicity correlates with the levels of covalent complexes (9). For
camptothecin-induced topoisomerase I-DNA covalent complexes, processes
such as DNA replication can convert the (reversible) protein-DNA lesion
into an irreversible lesion (38). DNA replication also can convert the
single-strand break formed by topoisomerase I into a double-strand
break (39). Both the protein-DNA "adduct" and the generation of
secondary double-strand breaks could contribute to camptothecin
cytotoxicity. It is therefore plausible that other agents that increase
the level of topoisomerase I-cleavable complexes are likely to be cytotoxic by the same mechanisms. In this paper, we demonstrate that in
the presence of DNA damage, topoisomerase I also forms a stable
covalent complex similar to the cleavable complexes formed in the
presence of the anti-cancer drug camptothecin and that these covalent
complexes enhance the cytotoxicity of DNA damage.
Several trivial explanations for the hypersensitivity of cells
expressing topoisomerase I to DNA-damaging agents can be readily excluded. Although cells overexpressing topoisomerase I grow more slowly than cells expressing normal levels of this enzyme, cells expressing a catalytically dead topoisomerase I also grow more slowly
than wild type cells, but those cells are not hypersensitive to
DNA-damaging agents. Since both yeast and human topoisomerase I
expression leads to drug hypersensitivity, the sensitization to
DNA-damaging agents does not arise from either a peculiar property of
one of the enzymes or from the expression of a heterologous enzyme.
Using two different assays we have established that yeast topoisomerase
I can be efficiently trapped on UV-damaged DNA. First we used the
potassium/SDS assay, which was applied to measure protein DNA-covalent
complexes trapped by topoisomerase poisons both in vivo and
in vitro (40-42). Second we directly examined the levels of
protein-DNA complexes using an electrophoretic mobility shift assay.
Both assays gave quantitatively similar results when similar samples
were examined. The potassium/SDS assay indicated a 3-fold increase in
covalent complexes with 1000 J/m2 UV versus
~5-fold with the mobility shift assay. A potential advantage of the
mobility shift assay is the ability to examine DNA with several
different types of DNA damage. The shortcoming of the potassium SDS
assay is the necessity of separately labeling each DNA that has a
different type or amount of DNA damage.
Other recent studies also indicate that DNA damage is able to stabilize
topoisomerase I-DNA covalent complexes in vitro. Pedrini and
co-workers (13) first showed that purified topoisomerase I stably
cleaves UV-damaged DNA at sites at or near UV damage (13). Pommier and
co-workers (44) also find that topoisomerase I can form a stable
covalent complex at the sites of several different specific DNA
lesions. Earlier results had shown that factors such as DNA curvature
could stimulate topoisomerase I cleavage (43) and that topoisomerase I
could act at sites of DNA breaks. Taken together, these results suggest
that topoisomerase I action can be altered by many different changes in
DNA structure.
If the normal reaction of topoisomerase I at sites of DNA damage did
not lead to further DNA damage, topoisomerase I could act as an
efficient sensor of DNA lesions. Because the enzyme acting at damage
results in a more complex lesion, the recognition of damage by
topoisomerase I does not seem to be useful for promoting cell survival.
Results described here that cells completely lacking topoisomerase I
are not more sensitive to DNA damage than cells with wild type levels
of the enzyme suggest that DNA damage recognition does not appear to be
a normal indispensable role for this enzyme.
Osheroff and co-workers (19) find that some types of DNA damage
can also lead to trapping of topoisomerase II on DNA. In their studies,
abasic sites greatly stimulated topoisomerase II cleavage of DNA. Since
we failed to observe decreased survival in cells overexpressing
topoisomerase II, the trapping of topoisomerase II by DNA damage either
occurs infrequently in vivo, or cells possess an efficient
system for preventing topoisomerase II-mediated DNA damage. In results
to be presented elsewhere, we have found that topoisomerase II levels
increase after DNA damage, which leads us to suggest that trapping of
topoisomerase II in vivo is a relatively infrequent event.
It may be relatively infrequent for two reasons. First, topoisomerase
II cleavage is strongly inhibited by some types of DNA damage such as
photoproducts induced by UV light. Second, the DNA repair systems that
recognize abasic sites may be much more efficient at binding to abasic
sites than topoisomerase II. If so, then cells lacking apurinic
endonucleases may become sensitive to topoisomerase II dosage. We are
currently testing this hypothesis.
How then do cells deal with the dangerous activity of topoisomerase I
when DNA is damaged? In mammalian cells, polyADP-ribose polymerase is
rapidly activated by DNA strand breaks (44). A major target for
poly(A)DP-ribose polymerase is topoisomerase I, and modification of
topoisomerase I by this enzyme inhibits topoisomerase activity (45,
46). Yeast cells apparently lack this enzyme, so some other pathway
must function to attenuate topoisomerase I activity after DNA damage.
The inactivation of topoisomerase I should occur fairly rapidly to
prevent the formation of covalent complexes at sites of damage. Either
covalent modification or targeted degradation could rapidly inactivate
topoisomerase I after DNA damage. At present we do not know whether
yeast topoisomerase I is rapidly degraded or whether other processes
are able to inhibit the activity of the enzyme. Liu and co-workers (47,
48) show that human topoisomerase I is modified by the proteins of the ubiquitin family including small ubiquitin-related modifier
after camptothecin treatment, suggesting that either degradation or inactivation of topoisomerase I can be part of the cell response to DNA
damage. However, the activity and altered stability of topoisomerase I
conjugated to small ubiquitin-related modifier remains to be
demonstrated (49). Also, down-regulation of topoisomerase I after
ionizing radiation has also been reported (50).
There are likely other pathways that control topoisomerase I after DNA
damage as well as pathways that can repair the DNA damage arising from
topoisomerase I. Nash and co-workers (51, 52) recently describe a yeast
protein that can disjoin topoisomerase I covalent complexes. Since DNA
damage and not just topoisomerase I poisons such as camptothecin are
able to trap topoisomerase I on DNA, the enzyme described by Nash
likely functions as one DNA repair system designed to deal with the
ability of topoisomerase I to generate covalent complexes at the sites
of damage.
Our results connect topoisomerase I to pathways of DNA damage repair
and DNA damage tolerance, but the connection we propose is not that
topoisomerases participate in repair but, rather, as an impediment to
accurate repair. It has also been found that overexpression of
topoisomerase I is able to increase nonhomologous integration of
transfected DNA in yeast (53). The nonhomologous integration could
arise from the action of topoisomerase at sites of endogenous DNA
damage. This may suggest that topoisomerase I could play a significant
role in genome destabilization after DNA damage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
by one-step gene disruption
(22). A top1 derivative of the CH335leu was constructed
by one-step gene disruption (23). The top1 disruption
removes the entire open reading frame of TOP1 and replaces
it with the yeast LEU2 gene. The resulting strain is termed
CH335top1. Both strains CH335 and CH335leu were
transfected with yCP50 or pGALyTOP1 (24). The strains carrying yCP50
served as vector controls, whereas strains with pGALyTOP1, which
expresses yeast TOP1 under the control of the yeast
GAL1 promoter, were used for experiments where yeast
TOP1 was overexpressed. Overexpression of human
TOP1 was accomplished using the vector pGALhTOP1 (25), and
expression of Escherichia coli topoisomerase I used the
vector pGALECTOP1 (26). The three vectors for overexpressing type I topoisomerases in yeast were the gift of Dr. J. C. Wang. A vector carrying the Y727F mutation under the control of the yeast
GAL1 promoter was the gift of Dr. R. Sternglanz.
and TOP1+ cells,
the appropriate strains were grown in yeast
extract/peptone/dextrose/adenine (YPDA) medium as previously described
(10), exposed to MMS for 3 h, then plated to YPDA agar to
determine the surviving fraction. All determinations were performed
with at least three independent isolates; the results shown are the
means ± S.E.
80 °C.
(23). One unit of topoisomerase I is defined as
the amount of enzyme required to completely relax 400 ng of pUC18 in 30 min using relaxation assays as previously described (23). The
topoisomerase I preparation used in the experiments reported in this
paper had a specific activity of 20 units/µg of protein.
-32P]dCTP.
Unincorporated nucleotides were removed using Chroma-spin (CLONTECH) columns. The specific activities of
unirradiated and UV-irradiated DNA had similar specific activities, as
determined by scintillation counting, ~107 cpm/µg DNA.
The cleavage reactions of 50 µl contained 250 ng of DNA, 10 mM Tris-Cl, pH 7.5, 70 mM KCl, 5 mM
MgCl2, 0.1 mM EDTA, pH 8.0, 15 µg/ml
acetylated bovine serum albumin, and 8 units of yTOP1 protein. Where
indicated, samples also contained 50 µg/ml camptothecin. Reactions
were incubated at 30 °C for 10 min then terminated using 1 ml of
STOP buffer (1.25% SDS (w/v), 5 mM EDTA, 0.4 mg/ml salmon
sperm DNA). Then 0.25 ml of 325 mM KCl was added, and
samples were incubated at 65 °C for 10 min. The samples were placed
on ice for 10 min, then centrifuged in an Eppendorf microcentrifuge at
8000 rpm for 10 min. The supernatant was completely removed, and
samples were resuspended in 1 ml wash buffer (10 mM
Tris-Cl, pH 8.0, 100 mM KCl, 1 mM EDTA, 1 mg/ml
salmon sperm DNA). The samples were heated to 65 °C for 10 min then
held on ice for 10 min and centrifuged as before. The wash procedure
was carried out a total of three times. The final precipitate was
resuspended in 0.4 ml of H20. 0.1 ml was removed and added
to scintillation fluid, and radioactivity was determined by
scintillation counting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
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Fig. 1.
Overexpression of yeast topoisomerase I
confers hypersensitivity to MMS. Yeast cells pre-grown in
galactose were exposed to MMS in yeast synthetic growth medium
containing galactose. At the indicated times, aliquots were removed,
diluted, and plated to yeast synthetic medium (without uracil) to
determine the number of viable cells carrying the appropriate plasmid.
CH335 cells with yCP50 were exposed to 0% MMS (open
squares), 0.04% MMS (open circles), or 0.08% MMS
(open triangles). CH335 cells with pGALyTop1 were exposed to
0% MMS (closed squares), 0.04% MMS (closed
circles), or 0.08% MMS (closed triangles). Results
shown are the mean of three independent determinations. Error
bars indicate S.E., and symbols lacking error bars have
S.E. less than the size of the symbol.
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[in a new window]
Fig. 2.
Overexpression of yeast topoisomerase I
confers hypersensitivity to UV light and ionizing radiation.
Results shown are the mean of three independent determinations.
Error bars indicate S.E., symbols lacking error
bars have S.E. less than the size of the symbol. Panel
A shows the sensitivity to UV, and panel B shows
the sensitivity to ionizing radiation.
View larger version (18K):
[in a new window]
Fig. 3.
Overexpression of E. coli
topoisomerase I confers marginal hypersensitivity to MMS.
Yeast cells pre-grown in galactose were exposed to MMS in yeast
synthetic growth medium containing galactose using the same conditions
shown in Fig. 1. CH335 cells with yCP50 were exposed to 0% MMS
(open squares), 0.04% MMS (open circles), or
0.08% MMS (open triangles). CH335 cells with pGALECTopA
were exposed to 0% MMS (closed squares), 0.04% MMS
(closed circles), or 0.08% MMS (closed
triangles). Error bars indicate S.E.; symbols lacking error bars
have S.E. less than the size of the symbol.
View larger version (18K):
[in a new window]
Fig. 4.
Overexpression of an inactive yeast
topoisomerase I mutant does not increase sensitivity to MMS. Yeast
cells pre-grown in galactose were exposed to MMS in yeast synthetic
growth medium containing galactose using the same conditions shown in
Fig. 1. CH335 cells with yCP50 were exposed to 0% MMS (open
squares), 0.04% MMS (open circles), or 0.08% MMS
(open triangles). CH335 cells with pGALyTOP1Y727F were
exposed to 0% MMS (closed squares), 0.04% MMS
(closed circles), or 0.08% MMS (closed
triangles). Error bars indicate S.E.; symbols lacking
error bars have S.E. less than the size of the symbol.
View larger version (17K):
[in a new window]
Fig. 5.
Overexpression of yeast topoisomerase II does
not confer hypersensitivity to MMS. Yeast cells pre-grown in
galactose were exposed to MMS in yeast synthetic growth medium
containing galactose using the same conditions shown in Fig. 1. CH335
cells with yCP50 were exposed to 0% MMS (open squares),
0.04% MMS (open circles), or 0.08% MMS (open
triangles). CH335 cells with pDEDTOP2 were exposed to 0% MMS
(closed squares), 0.04% MMS (closed circles), or
0.08% MMS (closed triangles). Error bars
indicate S.E.; symbols lacking error bars have S.E. less
than the size of the symbol.
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[in a new window]
Fig. 6.
Overexpression of TOP1 does
not alter DNA damage-inducible gene expression. RNA from cells
carrying pGALyTOP1 was isolated from cells grown either in glucose
(therefore not overexpressing TOP1) or galactose (therefore
overexpressing TOP1). Cells under both conditions were also exposed to
MMS for either 1 or 3 h before RNA isolation. After
electrophoresis and transfer to a nylon membrane, the blot was probed
with a probe for the RAD54 gene. Specific conditions are indicated on
the figure.
Cells Do Not Show Enhanced Resistance to
MMS--
If cells with elevated levels of topoisomerase I are
hypersensitive to DNA-damaging agents, then it seemed plausible that cells completely lacking topoisomerase I have elevated resistance to
DNA damage. In addition, Muller and co-workers suggested that topoisomerase I plays a role in repair, based on the detection of
trapped topoisomerase I (36). We tested the sensitivity of isogenic
TOP1+ and top1 cells to MMS. For clarity,
only the results after a 3-h exposure to MMS are shown in Fig.
7. It is clear that the sensitivity of TOP1+ and top1 cells to MMS under these
conditions is the same. Similar sensitivities to UV and ionizing
radiation were also found (data not shown). Our results indicate that
wild type levels of topoisomerase I expression do not sensitize cells
to DNA damage nor does topoisomerase I play a detectable role in repair
for these DNA-damaging agents. These results agree with a previous
determination of the sensitivity of top1 cells to DNA
damage (37).
View larger version (18K):
[in a new window]
Fig. 7.
Yeast top1 cells have the same
sensitivity to MMS as cells expressing wild type levels of TOP1.
Yeast cells (either CH335leu or CH335top1) were pre-grown
in YPDA medium and then exposed to MMS. After a 3-h exposure, cells
were diluted and plated to YPDA plates to assess viability. Error
bars indicate S.E.; symbols lacking error bars have
S.E. less than the size of the symbol.
View larger version (26K):
[in a new window]
Fig. 8.
Yeast topoisomerase I can be trapped on
UV-damaged DNA. A modified potassium/SDS assay was applied to
measure levels of topoisomerase I-DNA covalent complexes on
unirradiated or UV-irradiated DNA. The UV-irradiated DNA was exposed to
1000 J/m2 UV light, then both unirradiated and irradiated
DNAs were used as substrates to measure protein-DNA covalent complexes.
The potassium/SDS assay was carried out as described under
"Experimental Procedures." The conditions are shown in the figure.
Each sample was analyzed in triplicate, and the results shown are the
mean ± S.E. Differences between all four samples were
statistically significant with p < 0.05 by two-way
t tests.
View larger version (42K):
[in a new window]
Fig. 9.
Detection of topoisomerase I-DNA covalent
complex formation on UV-damaged DNA by electrophoretic mobility shift
assay. An electrophoretic mobility shift assay was developed to
assess topoisomerase I-DNA covalent complex formation on UV-damaged
DNA. Detailed experimental conditions are presented in the text, and
the conditions for each lane are indicated on the figure.
Panel A shows an autoradiograph of the assay. Note that the
band corresponding to free DNA was cut out before hybridization to
reduce the amount of labeled DNA needed for hybridization. The bands
from the gel in A were quantitated using a
phosphorimager, and the resulting quantitiation of the
unirradiated and UV-irradiated samples are shown in panel B. CPT, camptothecin.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dr. Jerrylaine Walker for purifying yeast topoisomerase I and Mo Mehrpooya for help with cleavage assays. We also thank Drs. Rolf Sternglanz (SUNY, Stonybrook, NY), Connie Holm (University of California, San Diego, CA), and James C. Wang (Harvard University) for the gift of strains or plasmids.
| |
FOOTNOTES |
|---|
* This work was supported by National Institutes of Health Grant CA52814 (NCI), Core Grant CA21765, and by the American Lebanese Syrian Associated Charities (ALSAC).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.
To whom correspondence should be addressed: Molecular Pharmacology
Department, St. Jude Children's Research Hospital, 332 N. Lauderdale
St., Memphis TN 38105. Tel.: 901-495-2794; Fax: 901-521-1668; E-mail:
john.nitiss@stjude.org.
Published, JBC Papers in Press, May 15, 2001, DOI 10.1074/jbc.M102674200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: MMS, methyl methanesulfonate; YPDA medium, yeast extract/peptone/dextrose/adenine medium.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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