(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) ()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) ).
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 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.
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
HO at a concentration of 400 µg/ml.
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, 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.
Figure 2:
Topoisomerase I-mediated cleavable
complexes stimulation by UV-induced photoproducts. BamHI-cut
duplex pAT153 DNA (30 ng), 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.''
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/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.
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 [-
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) .
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: , 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.''
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, lanes 8-11).
Figure 4:
Comparison of topoisomerase I-mediated
cleavage sites stimulated by CPT and UV photodamage within the amp 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).
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 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.
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 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.
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 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.
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 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) .
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 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.
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.