From the Laboratory of Molecular Pharmacology,
Division of Basic Sciences, NCI, National Institutes of Health,
Bethesda, Maryland 20892-4255, the § Department of
Biochemistry and Molecular Pharmacology, Thomas Jefferson University,
Philadelphia, Pennsylvania 19107-5541, and the ¶ Department of
Chemistry, Massachusetts Institute of Technology,
Cambridge, Massachusetts 02139-4309
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ABSTRACT |
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We recently showed that abasic sites, uracil
mismatches, nicks, and gaps can trap DNA topoisomerase I (top1) when
these lesions are introduced in the vicinity of a top1 cleavage site
(Pourquier, P., Ueng, L.-M., Kohlhagen, G., Mazumder, A., Gupta, M.,
Kohn, K. W., and Pommier, Y. (1997) J. Biol.
Chem. 272, 7792-7796; Pourquier, P., Pilon, A. A.,
Kohlhagen, G., Mazumder, A., Sharma, A., and Pommier, Y. (1997)
J. Biol. Chem. 26441-26447). In this study, we
investigated the effects on top1 of an abundant base damage generated
by various oxidative stresses: 7,8-dihydro-8-oxoguanine (8-oxoG). Using
purified eukaryotic top1 and oligonucleotides containing the 8-oxoG
modification, we found a 3-7-fold increase in top1-mediated DNA
cleavage when 8-oxoG was present at the +1 or +2 position relative to
the cleavage site. Another oxidative lesion, 5-hydroxycytosine, also
enhanced top1 cleavage by 2-fold when incorporated at the +1 position
of the scissile strand. 8-oxoG at the +1 position enhanced noncovalent
top1 DNA binding and had no detectable effect on DNA religation or on
the incision step. top1 trapping by 8-oxoG was markedly enhanced when
asparagine adjacent to the catalytic tyrosine was mutated to histidine,
suggesting a direct interaction between this residue and the DNA major
groove immediately downstream from the top1 cleavage site. Altogether, these results demonstrate that oxidative base lesions can increase top1
binding to DNA and induce top1 cleavage complexes.
DNA is highly susceptible to reactive oxygen species in
vivo, especially hydroxyl radicals that are generated by various
forms of oxidative stress such as lipid peroxidation, products of
inflammation, cellular respiration, and nearly ultraviolet light (1,
2). Many oxidized bases have been identified in mammalian DNA (3). In vivo measurements indicate that oxidatively modified
bases range from a few hundred to 4 × 105/cell/day
depending on the technique used (4). Among these, 7,8-dihydro-8-oxoguanine
(8-oxoG)1 represents the most
abundant lesion with estimates of about 100,000 8-oxoG being formed and
excised on average per rat cell per day (5). A recent study comparing
different techniques reported a lesser but still formidable amount of
8-oxoG produced in young rat liver with a steady-state level of 0.04 adducts/105 G (6). Conversion of G to 8-oxoG results in
retention of three hydrogen bonds between 8-oxoG and C and hence does
not severely alter DNA architecture as determined by x-ray
crystallography (7) (see Fig. 1B). However, polymerases
involved in DNA replication selectively incorporate A opposite to
8-oxoG (8). In this case, 8-oxoG adopts the syn conformation, a
geometry favoring two hydrogen bonds with A (7, 9, 10) (see Fig.
1B). In the absence of repair, A misincorporation leads to
G-T transversions (11-14) that could be relevant to the carcinogenic
process. Pyrimidines also undergo oxidation in vivo and can
lead to potential mutagenic derivatives such as 5-hydrocycytosine
(5-ohC) (15, 16).
In Escherichia coli, oxidized bases are removed by specific
enzymes. 5-ohC is removed by endonuclease III and formamidopyrimidine DNA N-glycosylase also known as MutM (17, 18). MutM also
removes 8-oxoG paired to a C. When 8-oxoG is paired with A, the MutY
glycosylase excises the complementary strand A residue that has been
misincorporated by the polymerase (13). It has been proposed that the
abnormal DNA major groove hydrogen bonding properties of oxoG,
attributable specifically to the 8-oxo carbonyl, are responsible for
recognition by glutamine or asparagine residues of the glycosylases
(7). A number of cellular enzymes from yeast, Drosophila,
and humans are capable of recognizing 8-oxoG, including glycosylases
and the nucleotide excision repair complex (4, 19-23). The effect of
8-oxoG incorporation on other ubiquitous DNA processing enzymes is
still unknown.
In this study we have investigated the effect of 8-oxoG on eukaryotic
topoisomerase I (top1) using purified enzyme and oligonucleotides containing a unique top1 cleavage site (24, 25). DNA topoisomerases play a critical role in DNA metabolism by changing the topological state of DNA (26). Eukaryotic top1 is an essential (27, 28) and
ubiquitous nuclear enzyme (26, 29, 30). It catalyzes a reversible
transesterification reaction by forming a covalent 3'-phosphotyrosyl
bond between a DNA strand and an enzyme tyrosine residue
(Tyr723 for human top1) while leaving a 5'-hydroxyl
terminus downstream to the cleavage site (31). These reversible
cleavage complexes are the key catalytic intermediates that permit
relaxation of DNA supercoiling and recombination. top1 inhibitors such
as camptothecin and its derivatives inhibit the religation of the
top1 cleavage complexes (32, 33). DNA modifications such as uracil
mismatches, abasic sites, nicks, and vinyl chloride adducts can also
trap top1 cleavage complexes and in some cases lead to the formation of
irreversible cleavage complexes also referred to as "suicide complexes" (25, 34-38).
In this report, we show for the first time that 8-oxoG can promote
formation of top1 cleavage complexes by increasing top1 binding to DNA.
We also demonstrate the influence of the amino acid residue immediately
flanking the catalytic tyrosine by using various top1 mutants including
an asparagine-to-serine mutant that was previously found to induce
camptothecin resistance (39). These findings further extend the broad
variety of DNA lesions affecting top1 activity and provide novel
details on the interactions of specific residues of eukaryotic top1
with DNA.
Chemicals--
High pressure liquid chromatography purified
control and 8-oxoG-containing oligonucleotides were purchased from The
Midland Certified Reagent Company (Midland, TX). 5-ohC-containing
oligonucleotides (5' to 3': GATCTAAAAGACTT5-ohCGAAAAATTTTTAAAAAAGATC,
GATCTTTTTTAAAAATTTTTC5-ohCAAGTCTTTTAGATC, and
GATCTTTTTTAAAAATTTTT5-ohCCAAGTCTTTTAGATC) were synthesized as described
previously (18, 40). [ Oligonucleotide Labeling and Annealing Procedures--
3'
Labeling was performed using terminal deoxynucleotidyl transferase
(Life Technologies, Inc.) with [ top1 Purification--
Overexpression of the human recombinant
top1 was performed in Sf9 insect cells using a baculovirus virus
construct containing the top1 full-length cDNA (42). Nuclear
extracts were prepared from infected Sf9 cells 4 days after
infection as described previously (25) and kept at top1 Reactions--
DNA substrates (approximately 50 fmol/reaction) were incubated with yeast or human recombinant top1 or
nuclear extracts for 30 min at 25 °C in reaction buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 µg/ml
bovine serum albumin). Reactions were stopped by adding SDS (final
concentration, 0.5%). For reversal experiments, the SDS stop was
preceded by the addition of NaCl (0.5 M for 30 min at
25 °C for standard reactions or 0.35 M NaCl at 4 °C
for reversal kinetics).
Gel Electrophoresis and Analysis of Cleavage
Products--
Before loading of the electrophoresis, 3.3 volumes of
Maxam Gilbert loading buffer (98% formamide, 0.01 M EDTA,
1 mg/ml xylene cyanol, 1 mg/ml bromphenol blue) were added to reaction
mixtures. 16% denaturing polyacrylamide gels (7 M urea)
were run at 40 V/cm at 50 °C for 2-3 h and dried on 3MM Whatman
paper sheets. Imaging and quantitations were performed using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
DNA Binding Assay--
3'-Labeled DNA substrates (approximately
25 fmol/reaction) were incubated in the presence of increasing
concentrations of yeast top1Y727F mutant protein (final volume, 20 µl) in 50 mM Tris-HCl, pH 7.5 (47), and 50 ng of
double-stranded poly dI-dC DNA (Sigma) for 5 min at room temperature.
10 µl of 3× neutral loading buffer (10 mM Tris HCl, pH
7.5, 30% glycerol, 0.1% bromphenol blue) were added to each reaction
before loading into a 6% native polyacrylamide gel in 0.25× TBE.
Electrophoresis was for 2 h at 100 V in 0.25× TBE buffer. Imaging
and quantitations were performed using a PhosphorImager.
Induction of Reversible top1 Cleavage Complexes by 8-Oxoguanine
Incorporation at the +1 Position of a top1 Cleavage Site--
In this
study, we investigated the effects of the abundant oxidative lesion
8-oxoG on top1 activity and how CPT would affect top1-DNA interactions
under such conditions. For this purpose, we used oligonucleotides
derived from a Tetrahymena ribosomal DNA sequence (24)
containing a single top1 cleavage site that had been previously mutated
to maximize top1 sensitivity to camptothecin and its derivatives (25,
48) (Fig. 1A). We first
studied the effect of 8-oxoG incorporation at the +1 position of the
scissile strand (X in Fig. 1A) on top1-mediated
cleavage using purified recombinant human enzyme. Replacement of G by
8-oxoG at the +1 position enhanced cleavage 3-5-fold (Fig.
1C, compare lanes 2 and 7). The same
increase (approximately 6-fold) was observed when 8-oxoG was paired
with A as it would be after polymerase misincorporation (Fig.
1C, compare lanes 2 and 12). By
contrast, 8-oxoG at the +1 position of the nonscissile strand
(Y in Fig. 1A) did not affect top1-mediated DNA
cleavage (Fig. 1C, compare lanes 17 and
22). These results are consistent with our previous finding
that DNA alterations at this +1 position can trap top1 and increase DNA
cleavage (36, 38). Under normal conditions, top1 cleavage is
reversible, leading to religation of the DNA strand. The top1
cleavage/religation equilibrium can be shifted toward religation by
increasing the salt concentration (48). In the case of the
8-oxoG-containing oligonucleotides, top1 cleavage remained reversible
after the addition of 0.5 M NaCl (Fig. 1C, lanes C). Similar results were obtained when 5-ohC was used
as a modified base. When introduced at the +1 position of the scissile strand, 5-ohC enhanced top1 cleavage approximately 2-fold (Fig. 2B, compare lanes 2 and 7). However, 5-ohC failed to affect top1 activity when
it was incorporated at the +1 (or the +2) position of the nonscissile
strand opposite to G (Fig. 2C, compare lanes 17 and 22 to lane 12). Interestingly, both the 8- oxo and the 5-hydroxy group of the oxidized +1 base on the scissile
strand occupy the same region of the DNA major groove (Fig.
1B, middle panel, and Fig. 2A,
right panel). Together, these results indicate that presence
of an additional oxygen group in the DNA major groove on the +1 base of
the scissile strand selectively enhances the formation of top1 cleavage
complexes.
Because we previously hypothesized that CPT traps top1 cleavage
complexes by interacting with the +1 purine of the top1 cleavage site
(49), we tested whether the 8-oxoG and 5-ohC substitutions at the +1
position would affect the activity of CPT. Fig. 1C shows that the 8-oxoG modifications had no effect on top1 sensitivity to CPT
when 8-oxoG was paired to C (lane 9). Cleavage was also salt-reversible (compare lanes 9 and 10).
However, when 8-oxoG was paired to A, CPT was not able to further
enhance top1 cleavage (compare lanes 14 and 12). Fig.
2B also shows the lack of effect of 5-ohC on CPT activity
(lane 9). These results demonstrate the potency of
8-oxoguanine and 5-hydroxycytosine to trap top1 reversibly when present
3' to the top1 cleavage site on the scissile strand (+1 position).
Induction of top1 Cleavage Complexes by 8-Oxoguanine Incorporation
at the +2 or
CPT remained active at the pre-existing top1 cleavage site in the
oligonucleotide with 8-oxoG at positions +2 and Effects of 8-Oxoguanine on top1-mediated DNA Cleavage and
Religation--
To assess the manner by which 8-oxoG enhanced top1
cleavage, we first performed kinetic experiments with the substrates
shown in Fig. 1. Cleavage was enhanced in the 8-oxoG oligonucleotide after 1 min and reached a plateau at about 3-5-fold the cleavage level
measured in the control oligonucleotide (Fig.
4A). top1-mediated DNA
religation was studied at 0 °C after addition of 0.35 M
NaCl (Fig. 4B). Under these conditions, religation is slower
than at 37 °C and in the presence of 0.5 M NaCl (48). As
a comparison, we used the control oligonucleotide in the presence of
CPT, because enhancement of top1 cleavage by CPT is known to result
from inhibition of religation of top1 cleavage complexes (48). After
NaCl addition, cleavage was reversed in the case of 8-oxoG after 1 min,
whereas CPT-induced cleavage remained detectable for more than 1 h. When compared with the control oligonucleotide in the absence of
CPT, religation rates were comparable, as 80% of the cleavage
complexes reversed after 5 s (data not shown). Thus, 8-oxoG at the
+1 position does not appear to mimic the effect of CPT, e.g.
to inhibit top1-mediated DNA religation.
To investigate the effects of 8-oxoG on the induction of DNA cleavage
by top1, we used a partially single-stranded 19-mer oligonucleotide,
also referred to as suicide substrate, containing the 8-oxoG at the +1
position relative to the top1 cleavage site (Fig.
5). Once top1 becomes covalently attached
to the 3' DNA terminus, it releases a 5'-hydroxyl hexamer (35, 50). The lower strand was phosphorylated at its 5'-end to prevent intramolecular religation (41, 50). Thus, using this oligonucleotide, cleavage can be
studied under single turnover conditions (51). Fig. 5 shows that the
presence of 8-oxoG at the +1 position did not increase top1 cleavage
rate as compared with the control oligonucleotide. Cleavage appeared
even slightly slower in the case of the 8-oxoG-containing oligonucleotide (Fig. 5C). This result demonstrates that the increase in top1 cleavage complexes observed at equilibrium (such as in Fig.
1C) was not due to an increased rate of DNA cleavage.
8-Oxoguanine Enhances the Formation of Noncovalent top1-DNA
Complexes--
We next investigated the effect of 8-oxoG on the
formation of noncovalent top1-DNA complexes using a yeast top1 mutant
where the catalytic tyrosine 727 was mutated to phenylalanine
(top1Y727Fp). This catalytically inactive mutant cannot perform the
incision step but is still able to bind the top1 consensus sequence
(52, 53).4 Using
electrophoretic mobility shift assays, we found that increasing concentrations of top1Y727Fp produced an increase in noncovalent top1-DNA complexes (Fig. 6A).
More efficient top1Y727Fp binding was observed with the oligonucleotide
containing 8-oxoG rather than G at the +1 position (Fig. 6A,
compare lanes 2-5 between the G:C (control) and the
8-oxoG:C oligonucleotides)). Quantitation of the top1-DNA complexes
indicated about a 3-fold increase with the highest concentrations of
enzyme (Fig. 6A, lanes 5) and about 7-fold with
the lowest concentrations (Fig. 6A, lanes 2).
Incorporation of 5-ohC at the +1 position of the nonscissile strand,
however, did not enhance top1Y727Fp DNA binding under the same
conditions (Fig. 6B). Thus, enhancement of top1 cleavage by
incorporation of 8-oxoG appears related to enhanced binding of top1 to
the 8-oxoG-containing DNA.
Enhanced top1 Cleavage Complex by 8-Oxoguanine Is Influenced by the
top1 Amino Acid Residue Immediately Flanking the Catalytic
Tyrosine--
Eukaryotic top1s vary in size across species but share
highly conserved amino acids sequences especially in the vicinity of the catalytic tyrosine in the carboxyl terminus region (29) (Fig.
7A). We investigated the
contribution of the asparagine immediately flanking the catalytic
tyrosine for the enhancement of top1 cleavage complex by 8-oxoguanine.
For this purpose, we used three different yeast top1 mutants where
asparagine 726 was mutated to histidine, aspartic acid, or serine. Each
of the partially purified mutant proteins was tested with the control
oligonucleotide and with oligonucleotides containing an 8-oxoG at the
+1 or the +2 position (Fig. 7B). With wild-type top1 and the
asparagine to serine mutant protein, cleavage complexes were almost
undetectable with the control oligonucleotide (G:C at +1 position). In
contrast, when asparagine was mutated to aspartic acid or histidine,
top1 cleavage complexes were enhanced significantly, although the
overall percentage of DNA cleavage was below 5% (Fig. 7C).
Incorporation of 8-oxoG at the +1 or +2 positions led to a marked
increase in top1-mediated cleavage for wild-type yeast top1, which
confirms our previous findings using the human recombinant enzyme (Fig. 1C). Enhancement of cleavage was also observed when the
asparagine was mutated to histidine or serine (Fig. 7, B and
C). When 8-oxoG was incorporated at the +2 position of the
scissile strand, only the histidine mutant led to marked enhancement of
top1-induced DNA cleavage (approximately 13-fold increase as compared
with the control oligonucleotide). Thus, replacement of the asparagine immediately adjacent to the catalytic tyrosine of top1 by histidine enhances DNA cleavage in 8-oxoG-containing oligonucleotides.
This study shows for the first time that 8-oxoG, which is one of
the most abundant oxidative DNA lesions in mammalian cells (5), can
induce the formation of reversible top1-linked DNA single-stranded
breaks. In general, an increase of top1-mediated cleavage complexes
results from a shift in the cleavage/religation equilibrium. Until the
present study, enhanced top1 cleavage by DNA damages (base mismatches,
abasic sites, or vinyl chloride adducts; Refs. 36 and 38) was found to
result from inhibition of religation, as in the case of CPT (30, 32,
54). By contrast, in this study we found that 8-oxoG paired with C at
the +1 position enhanced top1 cleavage complexes by increasing the
binding of top1 to DNA. We also found that oxidation of cytosine +1 on
the scissile strand (5-ohC, another oxidative damage found to be
mutagenic in vivo; Refs. 15 and 55) similarly enhanced
top1-mediated DNA cleavage. This effect on top1 is remarkably specific
because we did not detect any significant change in cleavage or binding when 8-oxoguanine or 5-hydroxycytosine were incorporated at the +1
position on the nonscissile strand. Because replacement of G by 8-oxoG
in DNA does not change base pairing or overall DNA structure (7),
increased top1 binding is probably due to the presence of the
nucleophilic 8-oxo group in the vicinity of the cleaved strand in the
DNA major groove.
As shown in Fig. 1B, oxidation of G converts the 7 position
from hydrogen bond acceptor to donor and the 8 position from a weak
hydrogen bond donor to a strong hydrogen acceptor, O8 (7). Hydrogen
bond donor/acceptors within the DNA major groove are probably essential
for recognition by DNA repair enzymes such as the DNA glycosylase, MutM
(7). Furthermore, because the hydroxy group of 5-ohC and the 8-oxo
group of 8-oxoG are occupying approximately the same position in the
DNA major groove and because both enhance top1 cleavage complexes, it
is plausible that they exert their activity by the same mechanism. We
propose that the differential arrangement of hydrogen bond
donors/acceptors within the major groove enhances top1 binding to
8-oxoG or 5-ohC-containing DNA. Based on the recently published
crystallographic structure of a top1-DNA complex (56, 57), asparagine
722 in human top1 (and the corresponding Asn726 in yeast
Saccharomyces cerevisiae; Fig. 7A) is a plausible
hydrogen bond donor for the O8 of 8-oxoguanine. Asparagine 722 is
pointed roughly in line with the +1 base on the scissile strand (57). Changing asparagine 722 to histidine would probably enhance a potential
interaction with 8-oxoG because the 5-membered ring would probably
bring the amino group even closer to the oxygen, which would be
covalently bonded to the carbon 8 of
guanine.5 It is also possible
that the asparagine to histidine mutation increases the reactivity of
the adjacent catalytic tyrosine.
A frequent in vivo DNA modification is the incorporation of
an A opposite 8-oxoG by DNA polymerases. This lesion is mutagenic. top1-mediated DNA cleavage was further enhanced when 8-oxoG was paired
to A at the +1 position. 8-oxoG:A base pair is in an
syn-anti-conformation, resulting in the protrusion of the 8-oxo group
in the minor grove of the DNA and altered hydrogen bonding (Ref. 7 and
Fig. 1B). Moreover, 8-oxoG:A base pairing was able to
inhibit top1-mediated religation in the absence of CPT, whereas
8-oxoG:C could not (data not shown). We previously reported that DNA
mismatches at the +1 position of a top1 cleavage site reversibly
enhance top1-mediated cleavage complexes (36, 58). Thus, in the case of
the 8-oxoG:A, we propose that DNA structural distortions and inhibition
of religation contribute to top1 trapping.
CPT has been shown to interact with the +1 base immediately downstream
of the top1 cleavage site (49). This interaction has been suggested to
inhibit DNA religation by top1 and thus to trap the top1 cleavage
complexes. We found that CPT remained inhibitory when 8-oxoG was paired
with C at the +1 position. However, the CPT activity was markedly
decreased when 8-oxoG was paired to A at this same position. We
previously reported such a decrease of CPT activity in the case of
mismatches or abasic sites at this +1 position, whereas wobble base
pairing such as that of G:U did not reduce CPT activity (36). These
results suggest the importance of base pairing at the +1 position for
CPT activity. These results are also consistent with the selective
interaction of camptothecin at the top1-DNA interface with the +1 base pair.
It is also interesting that the top1 trapping effect of 8-oxoG was
observed with crude nuclear extracts from leukemia cells (data not
shown). This result suggests that top1 can recognize the 8-oxoG
modifications in the presence of other nuclear proteins and can
efficiently compete for binding. Induction of top1 cleavage complexes
by oxidized bases could potentially lead to DNA damage and cell death
(as in the case of CPT) or possibly to recombination and mutagenic
effects. The potential role that specific DNA repair pathways play in
the processing of these lesions has yet to be established.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]Cordycepin 5'-triphosphate
was purchased from NEN Life Science Products, and polyacrylamide from
Bio-Rad. Camptothecin (CPT) was provided by Drs. Wani and Wall
(Research Triangle Institute, Research Triangle Park, NC).
10-mM aliquots of CPT were stored at
20 °C, thawed,
and diluted to 1 mM in Me2SO just before use.
-32P]cordycepin as
described previously (41). Labeling mixtures were subsequently
centrifuged through a G25 Sephadex column to remove excess
unincorporated nucleotide. Radiolabeled single-stranded DNA
oligonucleotides were annealed to the same concentration of unlabeled
complementary strand in 1× annealing buffer (10 mM
Tris-HCl, pH 7.8, 100 mM NaCl, 1 mM EDTA).
Annealing mixtures were heated to 95 °C and slowly chilled overnight
to room temperature.
80 °C before
used. Human top1 was purified from nuclear extracts using nickel
nitrilotriacetic acid-agarose beads (Qiagen, Santa Clarita, CA). 50 µl of beads were placed into microcentrifuge tubes and washed three
times with 1 ml of wash buffer (50 mM Hepes, pH 7, 0.5 mM dithiothreitol, 10 mM MgCl2, 3 mM MnCl2, 50 mM KCl). 500 µl of
nuclear extract were incubated with beads for 30 min at 4 °C and
washed two times with 300 µl of the wash buffer. Endogenous top1 was
eluted with wash buffer containing 40 mM imidazole. Human
recombinant top1 was eluted with wash buffer containing 300 mM imidazole and dialyzed 3 h with 30 mM
potassium phosphate buffer, pH 7, 0.1 mM EDTA, 5 mM dithiothreitol. Bovine serum albumin (0.2 mg/ml) and
glycerol (30% v/v) were added to the purified top1, and aliquots were
stored at
20 °C. Yeast top1 extracts were performed as described
previously. Proteins have been corrected for concentration by Western
blotting, and the standard catalytic activity of all mutants was
checked in standard assays. A baculovirus construct expressing the
yeast mutant, top1Y727F, was constructed from plasmid YCpGAL1-top1Y727F (43) using the Bac-to-Bac baculovirus expression system from Life
Technologies, Inc. Purification of recombinant yeast top1Y727F protein
from infected Sf9 insect cells was essentially as described (44), except for a final phosphocellulose chromatographic step in place
of Mono-S.2 Yeast wild-type
top1 and mutant proteins in which Asn726 was mutated to
serine, histidine, or
aspartate3 were partially
purified from galactose-induced yeast cultures as described (45, 46).
top1 protein concentrations were corrected in Western blots, and the
catalytic activities of all the mutant proteins were assessed in a
plasmid DNA relaxation assay (45, 46).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Selective enhancement of top1 cleavage by
8-oxoG incorporation at the +1 position of the scissile strand of an
oligonucleotide containing a single top1 cleavage site.
A, modified Tetrahymena hexadecameric rDNA
sequence with a strong top1 cleavage site indicated by the
arrowhead labeled with [32P]cordycepin
(*A) at the 3' terminus of the scissile (upper)
strand. Oligonucleotides were synthesized with either guanine
(G) or 8-oxoG at the +1 position of the scissile strand (in
bold type). Cleavage at the indicated site
(arrowhead) yields a 23-mer product. B, schematic
representation of normal G:C base pairing, 8-oxoG:C base pair and
8-oxoG:A base pair resulting from misincorporation of A opposite to
8-oxoG by DNA polymerase. Dashed lines represent hydrogen
bonds, and the 8-oxo group of the 8-oxoG is circled.
Open arrows indicate hydrogen bond acceptors, and
filled arrows indicate hydrogen bond donors. C,
oligonucleotides with indicated base pairing at the +1 position
relative to the top1 cleavage site were used as follows: lanes
A, DNA alone; lanes B and C, + top1;
lanes D and E, + top1 + 10 µM CPT.
Reactions were performed at 25 °C for 30 min and stopped either
immediately with 0.5% SDS (lanes B and D) or
first incubated with 0.5 M NaCl (final
concentration) for an additional hour at 25 °C before
addition of 0.5% SDS (lanes C and E).
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Fig. 2.
Enhancement of top1 cleavage by 5-ohC
incorporation at the +1 position of the scissile strand and lack of
enhancement after incorporation at the +1 and +2 positions of the
nonscissile strand. A, schematic representation of C:G
and 5-ohC:G base pairing. Dashed lines represent hydrogen
bonds. B, control oligonucleotide (C:G at the +1 position)
or oligonucleotide with a 5-ohC at the +1 position of the scissile
strand (5-ohC:G) (see Fig. 1A) were reacted as follows:
lanes A, DNA alone; lanes B and C, + top1; lanes D and E, + top1 + 10 µM
CPT. Reactions were performed at 25 °C for 30 min and stopped either
immediately with 0.5% SDS (lanes B and D) or
first with 0.5 M NaCl (final concentration) for an
additional hour at 25 °C before addition of 0.5% SDS (lanes
C and E). C, control oligonucleotide (G:C at
the +1 position) or oligonucleotides with a 5-ohC at the +1 or +2
position of the nonscissile strand (G:5-ohC) were reacted as described
in B.
3 Position of a top1 Cleavage Site--
We next studied
the influence of the position of the 8-oxoG by replacing the guanines
present in the immediate vicinity of the top1 site (Fig.
3A). 8-oxoG at the +2 position
on the scissile strand also led to reversible top1 trapping (Fig.
3B), and top1 cleavage was increased to approximately the
same extent (4-6-fold) as for the 8-oxoG at the +1 position (Fig.
1C). By contrast, incorporation of 5-ohC at the +2 position
of the nonscissile strand did not increase the formation of top1
cleavage complexes (Fig. 2C, compare lanes 12 and
22). When the guanine at the
3 position of the nonscissile strand (lower strand) was replaced by 8-oxoG, cleavage at the normal
site remained detectable (Fig. 3B). However, a new cleavage site was observed two bases upstream (Fig. 3B, open
arrowhead). This observation is consistent with our previous
studies showing that the presence of uracil, abasic site, or of a nick
at the
2 position of the nonscissile strand all induced this new top1 cleavage upstream to the normal cleavage site (25, 36). It is
interesting to note that the 8-oxoG was incorporated at position
1
relative to the new cleavage site. We also tested the effect of this
8-oxoG incorporation at the
3 position of the nonscissile strand on
possible top1-induced cleavage on the lower strand and did not detect
any cleavage of this strand (data not shown).
View larger version (52K):
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Fig. 3.
Induction of reversible top1 cleavage
complexes by 8-oxoG incorporated at other positions flanking the top1
site. A, sequence of the central portion of 3'
end-labeled oligonucleotides (*) (see Fig. 1A for complete
sequence) containing 8-oxoG (G=O) at the +2 position of the scissile
strand (right) or at the 3 position of the lower strand
(middle). Filled and open arrowheads,
cleavage site generating the 23- and 25-mer products, respectively.
B, reactions were: lanes A, DNA alone;
lanes B and C, + top1; lanes D and
E, + top1 + 10 µM CPT. Reactions were
incubated at 25 °C for 30 min and stopped either immediately with
0.5% SDS (lanes B and D), or first with 0.5 M NaCl (final concentration) for an additional hour at
25 °C before addition of 0.5% SDS (lanes C and
E). Numbers to the right indicate product size
(in nucleotides).
3 (compare lanes D and B) and CPT-induced cleavage was
reversible (compare lanes E and D). However, CPT
did not increase top1-mediated cleavage at the new site (Fig.
3B, compare lanes 7 and 9). We have
previously described the same lack of CPT activity when an abasic site
was present at the
3 position (36). These results demonstrate
that 8-oxoG can enhance top1 cleavage when present at the +1 or +2 position relative to a pre-existing top1 site and that it can induce new top1 cleavages.
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Fig. 4.
Kinetics of formation and religation of top1
DNA cleavage complexes in an oligonucleotide with 8-oxoG incorporated
at the +1 position. 3' end-labeled oligonucleotides with either a
G (Control) or 8-oxoG paired to C (8-oxoG:C) at the +1
position (see Fig. 1, A and B) were reacted with
purified human top1. A, kinetics of cleavage induction.
Reactions were stopped with 0.5% SDS at the indicated times.
B, kinetics of religation were performed in the absence of
CPT for the 8-oxoG/C+1 oligonucleotide and in the presence of 10 µM CPT for the control oligonucleotide. DNA substrates
(lanes a) were incubated with purified top1 for 15 min at
25 °C. Reactions were either stopped with 0.5% SDS (lanes
0) or reversed at 4 °C by adding 0.35 M NaCl (final
concentration) before adding 0.5% SDS. Incubation times (in min) are
indicated above each lane. Quantitations are
plotted in the right panels.
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Fig. 5.
Influence of 8-oxoG incorporation on
top1-induced DNA cleavage using a suicide oligonucleotide.
A, structure of the 3'-labeled suicide oligonucleotide.
X represents the +1 base of the scissile strand: either G or
8-oxoG. The lower strand was phosphorylated (p)
at the 5' end to prevent recombination (41). B,
oligonucleotides (lanes 0) were incubated at 25 °C with
human top1 for 5 s, 10 s, 30 s, 1 min, 3 min, 5 min, 10 min, and 30 min (lanes 1-8, respectively). Reactions were
stopped with 0.5% SDS. C, quantitation of the data shown in
panel B.
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Fig. 6.
8-oxoG increases non covalent top1-DNA
complexes. A, approximately 25 femtomoles of 3'
end-labeled double-stranded 37-mer control (G:C) or 8-oxoG:C
oligonucleotides (see Fig. 1) were incubated with increasing
concentrations of yeast top1Y727Fp for 5 min at 25 °C.
Electromobility shift assay was then performed. Lanes 1-5
correspond to 0, 1.5, 3, 7.5, and 15 ng of purified yeast top1Y727Fp,
respectively. B, control (G:C) and G:5-ohC oligonucleotides
were incubated with increasing amount of purified yeast top1Y727Fp as
described in A. Electromobility shift assay was
performed under the same conditions as in A.
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Fig. 7.
Influence of the conserved top1 asparagine
adjacent to the catalytic tyrosine on top1 cleavage complexes induced
by 8-oxoG. A, primary sequence of the conserved region
of eukaryotic top1 surrounding the catalytic tyrosine
(Tyr723 for human, and Tyr727 for yeast
Saccharomyces cerevisiae). B, 3' end-labeled
oligonucleotides (see Figs. 1 and 3) were incubated with the same
concentration of different yeast top1 enzymes for 30 min at 25 °C,
and reactions were stopped with 0.5% SDS. Lanes 1, DNA
alone; lanes 2, wild-type top1 (N); lanes
3, N726D mutant (D); lanes 4, N726H mutant
(H); lanes 5, N726S mutant (S).
C, plots of the data presented in B.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. Lance Stewart and Kurt W. Kohn for discussions and suggestions during the course of these studies.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grants CA58755 (to M.-A. B.) and CA52127 (to J. M. E.).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: Bldg. 37, Rm.
5D02, NIH, Bethesda, MD 20892-4255. Fax: 301-402-0752; E-mail:
pommier{at}nih.gov.
2 J. Fertala and M.-A. Bjornsti, unpublished results.
3 J. Fertala, P. Pourquier, Y. Pommier, and M.-A. Bjornsti, submitted for publication.
4 Hann, C. L., Carlberg, A. L., and Bjornsti, M.-A. (1998) J. Biol. Chem. 273, 31519-31527.
5 L. Stewart, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: 8-oxoG, 8-oxoguanine; top1, DNA topoisomerase I; 5-ohC, 5-hydroxycytosine; CPT, camptothecin.
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REFERENCES |
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