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(Received for publication, October 19,
1995; and in revised form, January 6, 1996) From the
DNA topoisomerases have been proposed as the proteins involved
in the formation of the DNA-protein cross-links detected after
ultraviolet light (UV) irradiation of cellular DNA. This possibility
has been investigated by studying the effects of UV-induced DNA damage
on human DNA topoisomerase I action. UV lesions impaired the
enzyme's ability to relax negatively supercoiled DNA. Decreased
relaxation activity correlated with the stimulation of cleavable
complexes. Accumulation of cleavable complexes resulted from blockage
of the rejoining step of the cleavage-religation reaction. Mapping of
cleavage sites on the pAT153 genome indicated UV-induced cleavage at
discrete positions corresponding to sites stimulated also by the
topoisomerase I inhibitor camptothecin, except for one. Subsequent
analysis at nucleotide level within the sequence encompassing the
UV-specific cleavage site revealed the precise positions of sites
stimulated by camptothecin with respect to those specific for UV
irradiation. Interestingly, one of the UV-stimulated cleavage sites was
formed within a sequence that did not contain dimerized pyrimidines,
suggesting transmission of the distortion, caused by photodamage to
DNA, into the neighboring sequences. These results support the proposal
that DNA structural alterations induced by UV lesions can be sufficient
stimulus to induce cross-linking of topoisomerase I to cellular DNA.
Cyclobutane pyrimidine dimers (CPDs) ( DNA
topoisomerases are ubiquitous enzymes involved in a number of crucial
cellular processes, including replication, transcription, and
recombination. A relationship between cellular responses to DNA damage
and topoisomerases has been
proposed(6, 7, 8, 9) . The catalytic
cycle of DNA topoisomerases can be divided into several steps: 1)
enzyme-DNA binding; 2) DNA cleavage, resulting in a covalent attachment
between the protein and one terminus of the DNA nick; 3) DNA strand
passage; 4) poststrand passage DNA religation concerted with the enzyme
turnover (reviewed in (10) ). Treatment with strong protein
denaturants arrests the catalytic cycle after the cleavage event by
trapping the transient DNA-protein intermediate of step 2, termed
``cleavable complex.'' Several topoisomerase inhibitors
stabilize the cleavable complex by interfering with the strand passage
cleavage/religation equilibrium. Treatment of cellular DNA with these
inhibitors results in cryptic single and double strand breaks
associated with enzyme's covalent attachment to DNA (reviewed in (11) ). UV photoproducts cause alterations of the DNA
conformation that can affect the activity of DNA processing enzymes. It
has been shown that UV irradiation of the substrate inhibits digestion
of recognition sequences containing dimerizable pyrimidines by
restriction enzymes (12) . Moreover, the catalytic reaction of
bacterial DNA topoisomerase I (13, 14) and Drosophila melanogaster DNA topoisomerase II (15) is
inhibited by UV damage in the target DNA. The molecular mechanism by
which damage can affect the enzyme's DNA strand passage step is
unknown. However, it has been proposed that the inhibitory lesions may
be present in the enzyme's active site at the time of strand
passage (15) or that the helical distortion induced by
photodamage to DNA can slow down the diffusion of the helix through the
DNA-protein bridge(13) . In an effort to verify whether
eukaryotic DNA topoisomerases might be the proteins involved in the
formation of UV-induced DNA-protein cross-links, the effects of short
wave UV-induced photoproducts on the enzymatic activity of human DNA
topoisomerase I were investigated. Our results indicate that the
enzyme's relaxation reaction is inhibited by the presence of UV
damage in the substrate. Reduced relaxation activity correlated with
alteration of the cleavage/religation equilibrium of the reaction,
resulting in the stimulation of cleavable complexes. This observation
supports the notion that DNA topoisomerase I may be the protein
involved in UV-induced DNA-protein cross-linking of cellular DNA.
BamHI, ScaI, Asp700, PvuI, PstI, and EcoRI restriction endonucleases, calf
thymus intestinal alkaline phosphatase, and DNA 3`-end-labeling kit
were purchased from Boehringer Mannheim (Mannheim, Germany). SalI was obtained from Pharmacia (Upsala, Sweden). T Plasmid pAT153 DNA (3657 bp, Fig. 3B) was purified from stationary phase HB101 Escherichia coli cells grown in L-broth supplemented with 0.2%
casamino acids and 10 µg/ml tetracycline, by alkaline lysis method
as described(18) . Separation of the negatively supercoiled
form DNA (RFI) from the nicked form DNA (RFII) was obtained on a
NACS-37 column (Life Technologies, Inc.) as described
previously(19) . DNA was stored at -20 °C in
H
Figure 3:
Localization of topoisomerase I-mediated
DNA cleavages stimulated by UV damage on pAT153 genome. A, the BamHI/SalI pAT153 restriction fragment (3382 bp),
uniquely 5`-end-labeled at the BamHI site, was reacted with
topoisomerase I and analyzed as in the legend of Fig. 2. The
autoradiogram of a typical gel is shown. Lane 1, DNA alone; lane 2, DNA and topoisomerase I in the presence of 2.5
µM CPT; lane 3, DNA and topoisomerase I; lanes 4-8, topoisomerase I and DNA irradiated at 440,
880, 1320, 1750, and 2200 J/m
Figure 2:
Topoisomerase I-mediated cleavable
complexes stimulation by UV-induced photoproducts. BamHI-cut
duplex pAT153 DNA (30 ng),
The negative of the gel photograph was
scanned with Model GS-670 Densitometer (Bio-Rad) and peaks quantitated
by integration with Bio-Rad Molecular Analyst software. The velocity of
DNA relaxation was determined by quantitating the loss of supercoiled
DNA as a function of time. The loss of supercoiled DNA was calculated
as (A
Localization at nucleotide level of
topoisomerase I-mediated cleavage sites was obtained by linearization
of pAT153 DNA with PvuI or Asp700 restriction
enzymes. Protruding and blunt 3`-ends were labeled using terminal
deoxynucleotidyl transferase and [
When cleavage products were to be analyzed
by acrylamide gel electrophoresis, DNA was dissolved in 5 µl of
loading buffer (80% formamide, 1 mM EDTA, 0.1% xylene cyanol,
0.1% bromphenol blue), subsequently heated at 90 °C for 2 min prior
to electrophoresis through 5% acrylamide, 7.5 M urea gels
prepared in Tris borate buffer (89 mM Tris-HCl, pH 8.0, 89
mM boric acid, 2.5 mM EDTA). Electrophoresis was at
50 watts for 2-4 h; gels were visualized by autoradiography as
above.
Figure 1:
Time course of
DNA topoisomerase I-catalyzed DNA relaxation in the presence of
UV-induced photoproducts. A illustrates the percent relaxation
of plasmid pAT153 DNA irradiated at the following UV doses versus time:
Cleavage was studied
by reacting 5`-end-labeled EcoRI-cut pAT153 DNA with
topoisomerase I for 5 min at 37 °C; the transiently nicked
enzyme-DNA intermediate was trapped by addition of SDS and digestion
with proteinase K followed by separation of reaction products on
alkaline agarose gel and visualization by autoradiography. Cleavable
complex stimulation was evaluated by measuring the decrease of
full-length linear molecules as a function of UV dose. As shown in the
gel photograph (Fig. 2A), there is a continuous
decrease of uncleaved molecules with the increase of UV irradiation. As
summarized in the graph (Fig. 2B), where results were
plotted as residual uncleaved DNA versus UV dose, there is
linear dependence of global cleavage with increasing amount of
photoproducts. This finding clearly indicates that the presence of
UV-induced DNA lesions affects the DNA cleavage/religation equilibrium
by causing the accumulation of cleavable complexes. This effect is
analogous to that described for the specific topoisomerase I inhibitor
camptothecin (CPT)(22) , and for mismatches adjacent to a
topoisomerase I cleavage site(23) .
A uniquely
end-labeled fragment (generated by secondary digestion of
5`-end-labeled BamHI-cut pAT153 DNA with SalI) was
incubated with a concentration of DNA topoisomerase I that produced
only limited DNA cleavage (Fig. 3A, lane 3),
denatured in the presence of SDS and deproteinized with proteinase K.
Localization of strand breakage was obtained by size analysis of the
cleavage products separated by alkaline agarose gel electrophoresis in
parallel with size markers. In Fig. 3A, the cleavage
pattern of the labeled strand irradiated at increasing UV doses is
shown. UV-stimulated topoisomerase I-mediated cleavages appeared
nonrandomly distributed. Damage induced bands (lanes
4-8) corresponded to breaks at pre-existing topoisomerase I
sites (lane 3) or at CPT-induced sites (lane 2)
except for one site (indicated by arrow). Using two
different uniquely 5`-end-labeled fragments,
(5`-BamHI/SalI and 5`-ScaI/Asp700),
genomic localization of cleavage sites was obtained. The position of
these sites on the pAT153 map was determined by densitometric scanning
of the autoradiograms and computer analysis of six independent
experiments with an average standard error of ± 25 nucleotides (Fig. 3B). The break site uniquely stimulated by UV
damage (Fig. 3A, arrow) was at position 3134
(±11 bp) of the plasmid map. The same analysis performed on the
complementary strand did not reveal any breakage site stimulated only
by UV damage (data not shown).
The 752-bp EcoRI/PstI fragment (5`-end-labeled at the EcoRI restriction site) was incubated with topoisomerase I in
the presence of increasing amount of CPT or after irradiation at
increasing UV dose (Fig. 4). Cleavages with the enzyme alone are
limited (lanes 2 and 7), while several additional
cleavage sites were visible on UV-irradiated DNA (lanes
8-11) or in the presence of CPT (from 0.75 to 5.0
µM, lanes 3-6). Cleavage stimulation by CPT
appeared concentration-dependent (lanes 3-6). No major
change in band intensities and in cleavage pattern could be observed as
a consequence of increase in UV irradiation (from 880 up to 2200
J/m
Figure 4:
Comparison of topoisomerase I-mediated
cleavage sites stimulated by CPT and UV photodamage within the amp
Breakage sites within
this fragment were tentatively classified into three categories
according to the response elicited by UV or CPT. Topoisomerase I
cleavages at a sites were stimulated by UV damage and by CPT,
those at b sites only by CPT, and those at c sites
specifically by UV irradiation of the target DNA. Among the c sites, the c1 site corresponded to the uniquely
UV-stimulated site identified by alkaline agarose gel electrophoresis.
Interestingly, c3 and c4 sites were formed at some
distance from the major CPDs clusters (lane 12). To examine
whether c sites were UV-specific, we increased the molar ratio
of enzyme to DNA. We found that a 3-fold increase in the molar ratio of
enzyme to DNA resulted in very weak breakage at c1 and c2 sites on nonirradiated DNA. These sites appeared slightly
stimulated also by CPT. No effect was observed at the c3 and c4 sites even with more enzyme (data not shown). The cleavage
sites stimulated by UV damage (c and a sites) were mapped
at nucleotide level by running the reaction products on sequencing gels
in parallel with Maxam-Gilbert chemical degradation reactions and
PD-endonuclease digestion products of the same end-labeled fragment.
The SspI-PvuI pAT153 restriction fragment
(3`-end-labeled at the PvuI restriction site) was used to
characterize c1 and c2 sites and the EcoRI-XmnI fragment (3`-end-labeled at the XmnI restriction site) to study c3 and c4 sites. In Fig. 5, the portion of the gel containing the
topoisomerase I cleavage products at the a1, a2, and c2 sites, obtained after stimulation with UV damage (lane
6), with CPT (lane 7) or with CPT on an irradiated
substrate (lane 5), is shown. Control experiments, in which
DNA was irradiated and topoisomerase I was omitted (lane 3),
excluded the possibility that cleavages were caused by UV light. The
two a sites, previously identified as single bands (Fig. 4, lanes 8-11), appeared to be flanked by
two b sites that were named b1 and b2.
Interestingly, cleavage intensity at a1, a2, b1,
and b2 sites was markedly suppressed, when CPT stimulation was
performed on an irradiated substrate (compare lanes 5 and 7). Inspection of the nucleotide sequences at the 5` terminus
of the topoisomerase I cleavages showed the presence of CPDs on the
irradiated substrate (lane 1). This effect was not detected
for other b sites that were instead located within sequences
that did not contain photodamage in the scissible strand, suggesting
that the presence of CPDs in the sequences adjacent to the break sites
can interfere with CPT action (data not shown).
Figure 5:
DNA sequencing analysis of topoisomerase I
cleavage sites stimulated by CPT and by UV damage within the amp
Fig. 6summarizes the nucleotide sequences containing the
UV-stimulated sites, the positions of the UV-stimulated topoisomerase
I-mediated cleavage sites with respect to the distribution of CPDs and)
photoproducts and the relative frequency of CPDs. Comparisons of the
base sequences upstream and downstream from the UV-stimulated sites has
not revealed any apparent specific elements that could explain the
stabilization of cleavable complexes observed in the presence of UV
damage. Analysis of the relative distribution of UV lesions on the
scissible strand indicate a difference between a and c sites. All three a sites were positioned at the 5` side of
pyrimidines runs that have an high probability to dimerize. With the
exception of the c2 site, the c sites were formed within
sequences that have a very low probability to contain damage in the
scissible strand. This is particularly evident for the c3 site
located at least 10 bases from two dimerized thymines.
Figure 6:
Relative frequency of CPDs formation and
DNA sequences at sites of topoisomerase I cleavage stimulated by UV
damage. Mapping of topoisomerase I cleavage sites within the amp
Figure 7:
Dissociation kinetics of cleavable
complexes. Two parallel reactions (120 µl), containing EcoRI/PstI restriction fragment (112 ng),
5`-end-labeled at the EcoRI site, were incubated with 672
units of topoisomerase I at 37 °C for 5 min after irradiation with
1750 J/m
It
is noteworthy that the UV-stimulated break sites appeared to have two
dissociation rates; an initial rapid rate in the first min of
incubation was followed by a very slow rate. The basis for the
discontinuity in the decay curves is unclear but it might involve a
rapid inactivation of the enzyme during the kinetic analysis or an
inability to complete break religation. In the latter case, it might
depend from the position of photodamage with respect to the break
sites. In fact, the irradiated substrate is constituted by an
heterogeneous population with respect to the position where damage is
created. Thus, in the fraction of molecules, where damage was present
at some distance from cleavage sites, resealing may occur at a much
higher rate (initial rate), while in the fraction of molecules, where
photodamage were formed close to the break sites, religation may be
very slow or even prevented (slow rate). The latter explanation is
supported by the observation that among the complexes stimulated by UV
damage, there is a good correlation between the religation rate and the
relative position with respect to damage. For example, the a1 and a2 sites, located in the scissible strand close to a run
of thymines with high probability to dimerize, decayed very slowly. The c3 site, formed within a sequence without dimers, appeared
instead to be reclosed more rapidly and to a greater extent. Cyclobutane pyrimidine dimers and) photoproducts, the most
common DNA damage induced by exposing DNA to short wave UV radiation (1) , produce a small but significant local distortion of the
double helix that can interfere with the proper functioning of enzymes
acting on DNA, such as restriction endonucleases (12) and DNA
topoisomerases. We have reported previously the inhibition of
prokaryotic DNA topoisomerase I catalysis by UV damage in the target
substrate(13, 14) . Analogous effect has been
described for D. melanogaster DNA topoisomerase
II(15) . In the present study we report the consequences of UV
damage on the activity of human DNA topoisomerase I. Our results
indicate that the presence of UV photodamage in the irradiated
substrate inhibited topoisomerase I action under steady state
conditions. As determined by DNA relaxation assay, the initial rate of
topoisomerase I catalysis decreased by approximately 50% when Decreased relaxation
activity by UV photodamage correlated with an interference of the
enzyme's cleavage/religation equilibrium resulting in the
stabilization of the cleavable complex. Cleavable complexes
stimulation, measured as induction of single strand breaks in linear
substrates, increased linearly with the dose. UV-dependent enhancement
of topoisomerase I-mediated breakages appeared to be due to a reduction
in the closure rate of the broken complexes. Thus, as established for
camptothecin, the molecular mechanism by which UV damage inhibited DNA
topoisomerase I catalytic activity seems to depend on its effects on
the religation step. However, we cannot exclude the possibility that
the presence of photolesions in the passing strand at the time of
strand passage may slow down strand diffusion through the DNA-protein
bridge. Several authors have demonstrated that DNA topoisomerase I
acts at preferred sequences lacking a clear consensus and that
camptothecin has only minor effects on the enzyme's nucleotide
specificity(30, 31) . Initial low resolution mapping
of UV-stimulated cleavage sites on the pAT153 genome did not reveal
major differences with respect to those stimulated by camptothecin
except for one position in the promoter of the amp Numerous studies have shown that steric factors
determine the interaction of topoisomerase I active site with
DNA(35, 36, 37) . Cleaved sites are
characterized by a set of distinct local helical parameters
(twisting)(26, 38) , and cleavage efficiency is
modulated by stable or dynamic curvature of the DNA molecule
(writhing)(28, 38, 39) . Evidences that
photodimers cause alterations in DNA structure are offered by numerous
physico-chemical, biochemical, and modeling studies. Measurements of
the shift of phased A-tract multimers containing site-specific CPDs (40) and of changes in band pattern of UV-irradiated
topoisomers (41, 42) have shown that CPDs cause a
topological unwinding due to a combination of actual duplex unwinding
(twisting) and negative supercoiling (writhing) resulting from bending
of DNA(40) . Thus, we hypothesize that the alteration in the
twist and writhe consequent to damage formation can determine changes
in the structural context that may either dislocate the enzyme-DNA
interacting sites of few bases (c2 and c4 site) or drive
into the optimal conformation for topoisomerase I activity sequences
normally poorly (c1 site) or not recognized by the enzyme (c3 site). This effect may resemble that described in supercoiled DNA,
where the activation of new sites, not detected in relaxed DNA, has
been observed(29) . In this respect the c3 site, formed
in a sequence with very low content of pyrimidine dimers in the strands
surrounding the cleavage site, is of special interest. In fact, if the
above hypothesis is correct, at least in this case one has to assume
that CPD conformational changes are not only localized at the damaged
pyrimidines but may be propagated into neighboring sequences as
postulated by Pearlman et al.(43) . It is well
established in the literature (1) and visible in Fig. 6that the frequency of dimer formation varies at different
potential dimer sites. Therefore, at the UV dose employed in the
mapping experiments (Fig. 5), the irradiated fragments consist
of an heterogeneous population of molecules with respect to UV damage
frequency and position. Because of that, it is quite difficult to
assess which of the lesions, that can be formed in the sequences
surrounding the UV-stimulated sites, is effective in stimulating
cleavage. In this respect, it cannot be excluded the possibility that
also UV photodamage present on the complementary strand can determine a
structural variation that may be reflected on the scissible strand. Although camptothecin does not seem to change significantly the
breakage specificity of the enzyme in vitro, it has a greater
stabilizing effect on some breakage sites than on others (reviewed in (30) and (31) ). The enhancement breakage factor
inversely correlates with the reclosure rate(32) . UV-induced
cleavage appeared to have different characteristics. The enhancement
factor did not seem to vary significantly from one site to another. In
addition, cleavage frequency at the sites identified in the EcoRI/PstI pAT153 fragment did not seem to be UV
dose-dependent. Nevertheless, from the dose-response curve of overall
cleavages, measured with the full-length DNA, there was a linear
increase of cleavable complexes stimulation up to a dose that
introduced approximately 100 CPDs per pAT153 molecule. This
contradiction may be explained by assuming that as the UV dose
increases damage is formed also at less probable positions. This
results in additional deformation of DNA structure that activates many
(potential) cleavable sites. However, each of them, being formed in low
amount, is cleaved with a very low frequency so that they cannot be
seen as discrete bands but as an increased background (Fig. 3).
Furthermore, the religation rate, reduced with respect to that measured
with CPT, did not correlate with the extent of cleavage but rather with
the position with respect to UV photodimers. When CPDs were present
within the sequence surrounding the cleavage sites, the rejoining step
afterwards was very slow. This case is particularly evident for the a1 and a2 sites that are flanked on the 5` side of the
break site by runs of thymines with an high probability to dimerize.
These sites showed a much lower religation rate when stimulated by UV
with respect to CPT stimulation. This type of kinetic can be explained
in terms of the extent of misalignment that the two ends at the site of
the nick have acquired as result of the helical distortion imposed by
the damage. Analogous explanation has been proposed for topoisomerase I
cleavage at a mismatch when present in one enzyme's recognition
sequence(23) . The UV doses employed in this study exceed
the level of radiation to which cells commonly are exposed. However,
the presence of UV lesions in critically located topoisomerase I
recognition sites could have physiological consequences. DNA
topoisomerases have been proposed as the enzymes responsible for sister
chromatid exchange (44, 45) and chromosomal
aberrations(46) . Several lines of evidence suggest a role for
eukaryotic DNA topoisomerases in mediating ``illegitimate
recombination'' of genetic material (reviewed in (47) ).
The mechanistic link derives from the capacity of topoisomerase
interactive drugs to stimulate these forms of genotoxicity ( (48) and reviewed in (5) and (46) ). The
biochemical basis for such a function comes from the ability of
topoisomerases to mediate cleavage and religation in two half-reactions
separated by the cleavable complex that gives the enzymes the capacity
to catalyze intra- and intermolecular DNA transfer
reactions(36, 49) . Moreover, it has been speculated
that also the generation of chromosomal rearrangements by DNA damage
could derive from their effects on
topoisomerases(44, 50) . Distortion of the DNA
structure in the vicinity of unrepaired DNA damage might be sufficient
stimulus to alter the correct function of these enzymes such that
incorrect rejoining could happen. However, UV damage does not stimulate in vitro the topoisomerase II-mediated cleavable
complex(15) . This observation implies that type II enzymes are
unlikely to be the protein that mediate UV-induced cross-links and
consequently responsible for UV-induced chromosome rearrangements.
Conversely, our finding that UV damage interfered with the activity of
DNA topoisomerase I by stabilizing the DNA-protein intermediate
necessary for strand exchange, supports the notion that UV-stimulated
DNA-protein cross-links may be mediated by topoisomerase I and that
this cross-linking may account for at least part of the chromosomal
rearrangements induced by UV light.
Volume 271,
Number 12,
Issue of March 22, 1996 pp. 6978-6986
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)and)
photoproducts are the most prevalent lesions produced in DNA by
ultraviolet (UV) light(1) . However, pyrimidine dimers are not
the only photochemical effect of UV light on cellular DNA. It has been
shown that UV radiation induces also the formation of DNA-protein
cross-links(2, 3, 4) . Proteinase K treatment
abolishes the cross-linking effect and reveals the presence of cryptic
DNA strand breaks. Since this cross-linking is partially repaired, it
has been suggested that this non-dimer DNA damage may play an important
role in the biological effect of UV radiation(2, 4) .
DNA topoisomerases have been proposed as possible candidates for the
protein(s) involved in UV-induced DNA-protein
cross-linking(4) . This proposal is consistent with the
formation of transient single and double strand breaks during DNA
topoisomerase reactions, with covalent attachment of the enzymes to one
terminus of the DNA nick (reviewed in (5) ).
Materials
Human DNA topoisomerase I, purified
from HeLa cell nuclei according to Ishii et al.(16) ,
was diluted to the appropriate concentration in DNA topoisomerase I
diluent (40 mM Hepes, pH 8.0, 0.2 mg/ml bovine serum albumin,
2 mM dithiothreitol, 0.5 mM EDTA, pH 8.0, 40%
glycerol, 6% poly(ethylene glycol)). One unit of enzyme is defined as
the amount of topoisomerase I required to relax 50% of 0.25 µg of
pAT153 DNA after a 10-min incubation at 30 °C. Micrococcus
luteus PD-endonuclease was obtained by purification to the
CM-cellulose step according to Grafstrom et al.(17) . 
polynucleotide kinase was from New England BioLabs (Beverly, MA).
[-
P]ATP and
[
-P]ddATP were obtained from Amersham
(Buckinghamshire, United Kingdom). Maxam and Gilbert sequencing kit was
from Merck (Darmstadt, Germany). Low melting Sea Plaque agarose GTG was
obtained from FMC BioProducts (Rockland, ME) and Gelase enzyme from
Epicentre Technologies Inc. (Madison, WI). Camptothecin, lactone form
NSC 94600, was obtained from Sigma.
O at a concentration of 400 µg/ml.
, respectively. The positions
and sizes of coelectrophoresed marker DNAs and the corresponding
genomic positions (in base pairs) are shown on the left. The
site uniquely stimulated by UV photodamage is indicated by an arrow. B shows the approximate genomic positions of
topoisomerase I-mediated DNA cleavage sites on the amp
coding strand of plasmid DNA, obtained by computer analysis of
six independent experiments using BamHI/SalI and ScaI/Asp700 restriction fragments. Bold indicates the map position of the break site uniquely stimulated
by UV photoproducts.
P-end-labeled at the 5`
termini, was reacted with 250 units of topoisomerase I (lanes
2-7), treated with SDS-proteinase K and analyzed on alkaline
agarose gel. In A is shown the autoradiogram of a gel
containing the full-length molecules. Lane 1, DNA alone; lane 2, topoisomerase I and unirradiated DNA; lanes
3-7, topoisomerase I and DNA irradiated at 440, 880, 1320,
1750, and 2200 J/m
, respectively. B illustrates
the results plotted as residual uncleaved DNA versus UV dose
on the basis of densitometric analysis of autoradiograms from three
independent experiments. Error bars represent the standard
errors. Assay conditions and quantitation of uncleaved DNA were
described under ``Experimental
Procedures.''
Preparation of UV-irradiated DNA
Ultraviolet
light-irradiated DNA was prepared by exposure to 254-nm UV light from a
germicidal lamp at a fluence rate of 1.89 J m s
, as determined by a VLX ultraviolet
intensity meter (Vilbert Lourmat, Marne-La-Vallé,
France). The samples were kept on ice to avoid heating and evaporation.
Under the conditions employed for short wave UV irradiation,
approximately 80% of the photoproducts induced in DNA are CPDs. The
remaining 20% are predominantly) photoproducts (data not shown). The
maximal UV dose employed in the present study (2200 J/m
) is
expected to generate approximately 100 CPDs/plasmid DNA molecule.DNA Topoisomerase I-catalyzed Relaxation of Negatively
Supercoiled DNA
The topoisomerase I-catalyzed DNA relaxation
assay was carried out as follow: each reaction mixture (120 µl
total volume) contained 1.50 µg of negatively supercoiled pAT153
DNA, nonirradiated or irradiated at the indicated doses, 50 mM Tris-HCl, pH 7.5, 120 mM KCl, 10 mM MgCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, pH 8.0, and 4.2 units of topoisomerase I. The reaction
mixture was incubated at 30 °C; 20-µl aliquots were removed at
the indicated times and added to 5 µl of stop mix (final
concentration 10% glycerol, 0.005% bromphenol blue, 0.1% SDS). DNA
samples were analyzed by electrophoresis at 25 V for 16 h on a 1%
agarose gel containing ethidium bromide in Tris-phosphate buffer (0.08 M Tris-phosphate, 0.8 mM EDTA, 0.5 µg/ml ethidium
bromide). After electrophoresis, the gel was subjected to photolysis by
short wave UV light (254 nm) to nick all DNA samples. This was done to
normalize the intensity of the observed bands, since supercoiled DNA
and relaxed DNA are stained differently with ethidium bromide. After
photonicking, the gel was destained and photographed under
transilluminator at 300-nm UV light with Polaroid type 55
positive/negative film./A+A
)
100, where A is the area of the relaxed
DNA band (RFIV) and A is the area of the
supercoiled DNA band (RFI). This automatically corrects for loading
variations or for differences in transilluminator efficiency. Under the
conditions employed, the band intensity in the negative of the gel
photograph was directly proportional to the amount of DNA present. To
examine the initial reaction velocity, the loss of supercoiled DNA was
quantitated at the different time points of the kinetic reactions.
Initial rates were calculated by a least squares fit analysis.Preparation of End-labeled DNA Fragments and Mapping of
DNA Breaks
To map topoisomerase I-mediated cleavage sites at
genome level, plasmid pAT153 DNA (Fig. 3B) was first
linearized with BamHI (position 375 of the genome) or ScaI (position 3140 of the genome) restriction enzymes; then
the 5` DNA termini were dephosphorylated with calf thymus alkaline
phosphatase and labeled with [-
P]ATP and T4
polynucleotide kinase. When needed, the labeled DNAs were subjected to
a second enzyme digestion with SalI (position 650 of the
genome) or Asp700 (position 3257 of the genome), respectively.
The longer DNA fragments were isolated and purified by low melting
agarose gel and digestion with Gelase enzyme. DNA was purified by
phenol-chloroform extraction and ethanol precipitation(18) .
The positions of cleavage sites, after DNA separation on alkaline
agarose gels, were obtained by densitometric scanning of the
autoradiographies and computer analysis of the data, as described
above. DNA markers were run in the two outer lanes of all gels in order
to check the uniformity of DNA migration throughout the gels.
Regression curves of the logarithm of the fragment size (in base
pairs), as a function of the migration distance of each fragment from a
reference line, were determined for the DNA markers. Regression
coefficients were consistently near 0.99. Each autoradiography lane was
analyzed using the same reference line, and the size of each DNA
fragment, induced by topoisomerase I cleavage, was computed. Fragment
size determination was usually within 50 bp for a given fragment
analyzed in different gels.
-P]ddATP
according to Brash (20) and then digested with SspI or EcoRI enzymes, respectively. The two 3`-end-labeled fragments
(435 and 398 bp, respectively) were purified by 5% acrylamide gel
electrophoresis, followed by electroelution and ethanol precipitation.
Mapping of the phosphodiester bonds cleaved by topoisomerase I was
obtained by electrophoresis of the reaction products on sequencing gel
alongside with the Maxam and Gilbert sequence ladder of the same
fragment(18) .
DNA Topoisomerase I Cleavage Reactions
DNA
fragments were incubated in 20 mM Tris-HCl, pH 7.5, 100 mM KCl, 10 mM MgCl, 0.1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 1
mM EDTA, pH 8.0, with appropriate amount of purified human
topoisomerase I at 37 °C for 5 min. Cleavable complexes were
disrupted by addition of 1% SDS and proteinase K (100 µg/ml),
followed by incubation for 30 min at 37 °C. DNA was then phenol
extracted and ethanol-precipitated. When cleavage products were to be
analyzed by alkaline agarose gel electrophoresis, equal counts from
each samples were dissolved in 5 µl of HO, adjusted to
50 mM NaOH and 1 mM EDTA, and loaded into the wells
of a 1% alkaline agarose gel. Alkaline agarose gels were run at 2 V/cm
overnight and then dried. Dried gels were autoradiographed with
Hyperfilm-MP (Amersham).M. luteus PD-endonuclease Digestion and Piperidine
Treatment
Analysis of UV photoproducts was essentially as
described by Brash(20) . To detect CPDs, the uniquely labeled
DNA was treated with M. luteus PD-endonuclease, which cleaves
the glycosidic bond of the 5` cyclobutane dimer and the phosphodiester
bond 3` to the apyrimidinic site, leaving a free 3` hydroxyl
end(21) . The UV-irradiated DNA was incubated with saturating
amount of M. luteus PD-endonuclease in 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 mM NaCl (final
volume 20 µl), at 37 °C for 30 min. The reaction was stopped by
phenol extraction, and DNA was subsequently purified by ethanol
precipitation. To detect ) photoproducts, the labeled DNA was treated
with hot alkali to break the labile glycosidic bond of the 3`
pyrimidine photoproduct, subsequently leading to 
-elimination at
the apyrimidinic site(20) . The UV-irradiated DNA was dissolved
in 100 µl of 1 M piperidine (Fluka) and heated for 30 min
at 90 °C. DNA fragments were precipitated by addition of sodium
acetate, pH 5.2, to a final concentration of 0.3 M and 2.5
volumes of ethanol. The precipitates were washed twice with 75% ethanol
and dried in a Speedvac concentrator to remove traces of remaining
piperidine. The position of cleavages by M. luteus PD-endonuclease and by hot piperidine was estimated by running the
treated samples on DNA sequencing gel, alongside with the Maxam and
Gilbert sequence ladder of the same fragments. The relative frequency
of UV photoproduct formation was calculated from the area of the peaks
obtained by densitometric scans of the autoradiographies as above. DNA
cleavage was expressed in arbitrary units relative to the most intense
band. Since the relative rates of photodamage formation are not
modified in the range of doses employed(21) , the UV dose
chosen for this analysis was calculated so to produce an average of 1
CPD per DNA strand.
Effect of UV-induced Photoproducts on DNA Topoisomerase
I-catalyzed DNA Relaxation
A DNA relaxation assay was utilized
to examine the effects of UV-induced damage in the substrate on the
catalytic activity of human DNA topoisomerase I. This assay measured
the conversion of naturally supercoiled pAT153 DNA (RFI) into relaxed
covalently closed circular DNA (RFIV). RFI and RFIV DNAs were separated
from the nicked molecules (RFII) by inclusion of ethidium bromide in
the agarose gel. In this condition the relaxed topoisomers migrate as a
single band ahead of the RFI band allowing an accurate quantitation of
the reaction products by densitometric scanning of gel photographs. A
time course of relaxation was carried out with catalytic amount of
enzyme and pAT153 DNA molecules irradiated at increasing UV doses from
0 to 1750 J/m (Fig. 1, A and B).
Under these conditions, the relaxation velocity of supercoiled DNA was
linear for 20 min. Following a 20-min incubation, 0.7 unit of
topoisomerase I relaxed about 65% of nonirradiated pAT153 DNA. As shown
in Fig. 1C, the initial DNA relaxation rate linearly
decreased as the level of UV-dose increased. At the highest UV dose,
the initial velocity was reduced to about 20%. These results indicate a
dose-dependent inhibition of topoisomerase I activity by UV-induced
damage.
, no irradiation; , 438 J/m
; 
875 J/m; 
, 1312 J/m; half-filled
diamond, 1750 J/m. Results are the average of two
independent experiments. B shows an agarose gel of a DNA
relaxation assay. Plasmid DNA nonirradiated (lanes 1-5)
or irradiated with 1750 J/m (lanes 6-10) was
incubated with 0.7 unit of topoisomerase I for the following length of
time: lanes 1 and 6, 0 min; lanes 2 and 7, 5 min; lanes 3 and 8, 10 min; lanes 4 and 9, 15 min; lanes 5 and 10, 20 min. C shows the initial reaction velocities determined as
described under ``Experimental Procedures.'' The results,
obtained from data shown in A, were plotted as a percent
relaxation activity versus the UV dose to which plasmid
substrates were exposed. The relaxation activity of topoisomerase I in
the absence of UV-induced photoproducts was set to 100%. The average
standard errors were less than 5%. Assay conditions and densitometric
analysis of reaction products on negatives of agarose gel photographs
were as described under ``Experimental
Procedures.''
Effect of UV-induced Photoproducts on the DNA
Cleavage/Religation Equilibrium of Topoisomerase I
The catalytic
cycle of topoisomerase I can be divided into several discrete steps
involving DNA binding, DNA cleavage, DNA strand passage, religation of
the DNA break, and enzyme turnover. The cleavage reaction involves a
transient single-stranded break in the DNA backbone containing the
enzyme covalently bound at the 3` side of the cleaved strand. This
transient intermediate, termed cleavable complex, can be evidenced by
freezing the cleavage/religation equilibrium with strong protein
denaturants. In order to determine which specific reaction step(s) of
the normal catalytic cycle is impaired by UV light-induced lesions in
the target DNA, the effect of this damage on the DNA
cleavage/religation equilibrium was examined.Genomic Localization of Topoisomerase I-mediated DNA
Breaks Stimulated by UV Photoproducts in Plasmid pAT153
DNA
Eukaryotic topoisomerase I-mediated cleavages are
nonrandomly introduced in the DNA helix. Cleavage sites are
characterized by a weak sequence-specificity (24, 25) and a loose conformational consensus (26) with preference for curved DNA, be it stably (27, 28) or dynamically bent(29) . Although
CPT has only a minimal effect on the sequence selectivity of the enzyme
(reviewed in (30) and (31) ), the degree of
stimulation differs among sites(32) . Experimental and modeling
studies have shown that UV damage is characterized by a small but
significant deformation of the DNA double helix, which can affect
protein-DNA interaction(12) . Thus, it is possible to envisage
that the modifications of the helical parameters produced by UV
photoproducts may interfere with the sequence specificity of
topoisomerase I-mediated cleavage. Therefore, it is of interest to
study topoisomerase I cleavage specificity on UV-irradiated DNA and to
compare this specificity with that described for CPT.Higher Resolution Analysis of Cleavage Site
Specificity
The influence of UV damage on the cleavage
properties of human topoisomerase I was further characterized by
analysis, on denaturing acrylamide gel, of the break sites previously
mapped within the region encompassing the site preferentially
stimulated by UV photolesions. The position of UV-induced breakages was
compared with the position of breaks stimulated by CPT and with CPDs
distribution, obtained by running in parallel the same UV-irradiated
fragment digested with the cyclobutane dimer-specific endonuclease from M. luteus(21) ., lanes 8-11).
gene. The EcoRI/PstI restriction
fragment (752 bp, genomic positions 3655-2903), spanning the
region containing the uniquely UV-stimulated site, 5`-end-labeled at
the EcoRI terminus (16 ng), was reacted with 100 units of
topoisomerase I after irradiation at increasing UV doses or in the
presence of increasing amount of CPT. Reaction products were processed
and analyzed on acrylamide gel as described under ``Experimental
Procedures.'' Lane 1, DNA alone; lanes 2 and 7, DNA and topoisomerase I; lanes 3-6, DNA and
topoisomerase I with 0.75, 1.25, 2.50, and 5.0 µM CPT,
respectively; lanes 8-11, topoisomerase I and DNA
irradiated at 880, 1320, 1750, and 2200 J/m, respectively; lane 12, UV-irradiated DNA (220 J/m) digested with
PD-endonuclease as described under ``Experimental
Procedures.'' Numbers on the left side correspond to the approximate genomic positions (in base pairs) in
the plasmid sequence obtained with appropriate size markers. The lowercase letters and the bars on the right indicate the positions of topoisomerase I-mediated cleavages
stimulated: by CPT and UV damage (a), by CPT only (b), and uniquely by
photodamage (c).
gene. The figure shows part of the sequencing
analysis of the PvuI/SspI restriction fragment (435
bp, genomic positions 3029-3464) 3`-end-labeled at the SspI site. DNA (2 ng) was reacted with 12 units of
topoisomerase I and processed as described under ``Experimental
Procedures.'' Reactions products were analyzed by denaturing
acrylamide gel electrophoresis. Maxam and Gilbert sequence ladders of
the same DNA substrate were analyzed in parallel with the topoisomerase
cleavage reaction products. Lane 1, UV-irradiated DNA (220
J/m) digested with PD-endonuclease; lane 2, DNA
alone; lane 3, DNA irradiated at 1750 J/m; lane 4, DNA and topoisomerase I; lane 5, DNA
irradiated at 1750 J/m and topoisomerase I in the presence
of 2.5 µM CPT; lane 6, topoisomerase I and DNA
irradiated at 1750 J/m; lane 7, DNA and
topoisomerase I in the presence of 2.5 µM CPT.
gene was performed using uniquely
3`-end-labeled restriction fragments essentially as described in Fig. 5and under ``Experimental Procedures.'' The
position of the break sites is indicated by an arrow. The
distribution and relative frequency of CPDs (closed bars) and
the distribution of) photoproducts, surrounding the topoisomerase I
cleavage sites, were obtained as described under ``Experimental
Procedures''; the pyrimidines involved in the formation of)
photoproducts are underlined.
Dissociation Kinetics of Enzyme-DNA Complexes by
Heating
Cleavable complexes stimulation by camptothecin has been
shown to be reversed by several treatments such as elevated temperature (33, 34) . To investigate the mechanism of
topoisomerase I breakage stimulated by UV photodamage, the stability of
preformed UV- or CPT-induced cleavable complexes was measured. The
dissociation kinetics were followed by heating the reaction mixtures at
65 °C before treating samples with SDS. The electrophoretic
analysis of the resulting DNA fragments is shown in Fig. 7A. It is clear that the UV- and CPT-stimulated
cleavable complexes showed widely different sensitivities to heating.
The enzyme-DNA complexes formed in the presence of CPT (a and b sites) dissociated very rapidly, while complexes formed after
UV irradiation (a and c sites) decayed more slowly, with
broken complexes still detectable after 15 min of heating. To quantify
the differences, the residual cleavage, i.e. the cleavage
frequency at a given time normalized to the cleavage frequency obtained
in a sample from the same experimental series subjected to SDS
immediately before heating, was plotted as a function of the incubation
time at 65 °C (Fig. 7B). The residual breakage at
the CPT-stimulated a and b sites (shaded area)
was reduced to less than 1% after 1 min at 65 °C and cleavage was
not detected at later times. In contrast, the complexes formed at
UV-stimulated sites decayed more slowly and remained incomplete.
or in the presence of 2.5 µM CPT. The
reaction mixture was then heated to 65 °C, and aliquots (20 µl)
were withdrawn at various times after the treatment. Reaction products
were processed and analyzed by denaturing acrylamide gel
electrophoresis as described under ``Experimental
Procedures.'' A, lanes 2-7, samples containing
UV-irradiated DNA withdrawn at 0, 0.5, 1, 5, 10, and 15 min,
respectively; lanes 8-13, samples treated with CPT
withdrawn at the same times as lanes 2-7. In lane 1 is shown a control sample of DNA treated with topoisomerase I and
processed after 5 min at 37 °C. B, in each of the
experimental series, the cleavage frequency of the indicated cleavage
sites at any point were expressed as a percentage of the cleavage
frequency in the sample taken before heating. This percentage was
plotted against the time of sampling. The quantification is based on
densitometric scanning of the autoradiogram shown in A.
Residual cleavage frequency at a (dashed lines) and c (continuous lines) sites in the presence of UV damage; shaded area, residual cleavage frequency at a and b sites in
the presence of CPT.
40
CPDs were present per plasmid pAT153 molecule. This level of inhibition
was lower than that previously reported for the prokaryotic type I
topoisomerase from M. luteus(13) and eukaryotic type
II from D. melanogaster(15) .
gene. Subsequent localization at nucleotide level of the breakage
sites in the region encompassing the uniquely UV-stimulated site and in
the neighboring sequences was carried out and a comparison between the
UV-stimulated sites with those induced by camptothecin was done. Some
sites were uniquely stimulated by UV damage (c sites) and
appeared at some distance from CPT-stimulated sites except for one (c4 site). Most CPT-stimulated breakages were not stimulated
also by the presence of photoproducts (b sites) except for few
sites (a sites). The relative position of b sites with
respect to CPDs distribution did not reveal any special features that
could give some clues to understanding why they were not stimulated
also by UV damage. One exception is offered by the two b sites
flanking sites a1 and a2 where breakages at both sites
were within a thymines run that dimerized with high efficiency. In this
case, it is possible to speculate that the cyclobutane bonds between
the two thymines flanking the break at the b sites may affect
either the cleavage and/or the binding of the enzyme to the substrate.
This possibility is supported by the observation that cleavage
stimulation by CPT on an UV-irradiated substrate was severely reduced
at these two sites, while it was not affected at b sites formed
at sequences without dimers in the scissible strand. Also the neighbor a1 and a2 sites showed a marked reduction in cutting
frequency when cleavage was examined in the above condition, suggesting
that UV damage stimulation was predominant at these sites and that
CPT-stimulated cleavage was effective only on the fraction of molecules
without damage in the flanking sequences. However, an alternative
explanation to this latter observation can be formulated based on the
model of the camptothecin-topoisomerase I-DNA ternary complexes
proposed by Pommier (reviewed in (31) ). According to this
model, the planar ring of camptothecin should stuck with the base at
the 5` terminus of the DNA breaks within the cavity formed at the
topoisomerase I cleavage sites. Thus, the presence of UV lesions on the
5` terminus of the breaks produces conformational deformations that may
alter the camptothecin receptor sites and consequently affect the
action of the drug.
)
We thank Dr. G. Capranico for critical reading of the
manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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