[CANCER RESEARCH 58. 976-984. March 1, 1998]

Ultraviolet-induced DNA Damage Stimulates Topoisomerase I-DNA ComplexFormation in Vivo: Possible Relationship with DNA Repair1

Deepa Subramanian, Barry S. Rosenstein, and Mark T. Muller2

Department of Molecular Genetics, The Ohio State University; Columbus, Ohio 43210 ID. S.. M. T. M.I; ami Department of Radiation Oncology, Mount Sinai School of Medicine,The City University of New York, New York, New York 10029 ¡B.S. R.¡

ABSTRACT

An antibody-based method was used to examine genomic DNA cleavageby endogenous topoisomerases in living cells. The method quantifies cleav-

able (covalent) complex formation in vivo after exposure to topoisomerasepoisons, as reported previously (D. Subramanian et al., Cancer Res., 55:2097-2103, 1995). Unexpectedly, exposing cells to UVB irradiation stimulated endogenous topoisomerase I-DNA covalent complex formation byas much as 8-fold, even in the absence of drugs that stabilize the cleavablecomplex. Covalent complexes are not a result of nonspecific UV protein-DNA cross-linking; rather, they result from the enzymatic activity of

topoisomerase I on genomic DNA. Because the action of topoisomerase IIon genomic DNA was not affected by UVB exposure, the observationappears to be specific for type I. Topoisomerase I is rapidly mobilized ontothe genome (within 12 min after UVB exposure); however, topoisomeraseI polypeptide levels did not show a corresponding increase, suggesting thatpreexisting enzyme is being recruited to sites of DNA damage. Complexespersist up to 5 h post-UV exposure (concurrent with the period of active

DNA repair), and their formation is independent of S phase. Thesefindings can be partially explained by the fact that in vitro topoisomeraseI activity on UV-damaged DNA tends to favor formation of cleavage

complexes; thus, a higher yield of covalent complexes are detected at ornear cyclopyrimidine dimer lesions. Because repair-deficient cells are

additionally compromised in their ability to recruit topoisomerase I, adirect role for the enzyme in DNA excision repair process in vivo isproposed that may be related to the activity of the xeroderma pigmento-

siini complementation group D helicase. Finally, these results collectivelydemonstrate that topoisomerase I is a repair-proficient topoisomerase in

vivo.

lowing UV irradiation (6). These results are consistent with the ideathat DPC formation after UV irradiation represents some form ofrepair process or cellular response to DNA damage. Because topoisomerases represent a class of nuclear proteins that covalently bindgenomic DNA, their relationship with DPC formation has been examined.

Topoisomerases are enzymes that alter the topology of DNA bybreaking and resealing DNA. The catalytic cycle of the type I enzymeinvolves the following steps: DNA binding. DNA breakage, passageof the DNA through the enzyme-DNA gate, resealing DNA breaks,

and release of the enzyme from the binding site (for reviews see Refs.8-10). Topoisomerase activity has been implicated in DNA templat-ing processes, such as transcription (11-14), replication (15, 16), and,possibly, DNA repair (17-20). During the breakage step, an interme

diate, which consists of a covalent complex between the broken DNAand the active site tyrosine, is formed (21-23). Both topoisomerase I

and topoisomerase II form covalent bonds with DNA (concurrent withDNA breaks); therefore, either of these proteins may be responsiblefor the observed DPC formation following UVB irradiation.

To examine the involvement of topoisomerases in UVB-induced

DPCs, we used an in vivo assay (the ICT bioassay) to measuretopoisomerase activity on genomic DNA in intact cells followingUVB irradiation (24, 25). The results demonstrate that UVB specifically stimulates the formation of covalent complexes between topoisomerase I and DNA covalent complexes but not topoisomeraseII-DNA covalent complexes.

INTRODUCTION

The UV component of sunlight, particularly the UVB region (290-

320 nm), is responsible for the induction of most skin cancers (1).Through the action of DNA repair systems, the principal lesions[cyclobutane dimers and pyrimidine (6-4)pyrimidome photoproducts]are removed (2). A minor class of photoproducts, DPCs,3 which

increase with time after UV exposure, are also produced (3-6). TheseDPCs are accompanied by the formation of single-strand breaks in

DNA (3, 7). Evidence supporting the biological role of this processwas obtained through the isolation of a mutant cell line, which washypersensitive to solar UV wavelengths and deficient in DPC andsingle-strand break formation (5). In addition, two cell lines obtained

from systemic lupus erythematosus patients were hypersensitive tosimulated sunlight and displayed a deficiency in the formation ofDPCs. A different systemic lupus erythematosus cell line, exhibitingnormal UV resistance, showed normal levels of DPC formation fol-

Received 6/25/97: accepted 1/2/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by NIH Grants CAI6058. CA63185. and CA63653 (to

M. T. M.).2 To whom requests for reprints should be addressed, at Department of Molecular

Genetics. The Ohio State University. 484 West 12th Avenue, Columbus. OH 43210.Phone: (614)292-1914; Fax: (614)292-4702; E-mail: muller.2@osu.edu.

' The abbreviations used are: DPC. DNA-protein cross-link; ICT, in vivo complex of

topoisomerase; XPA and XPD, xeroderma pigmentosum complementation groups A andD, respectively; CPT, camptothecin; CPD, cyclobutane pyrimidine dimer.

MATERIALS AND METHODS

Reagents. Adherent HeLa cells were cultured in 75-cnr flasks (Corning) inCO2-independent medium (Life Technologies, Inc.) supplemented with 10%defined, iron-supplemented bovine calf serum (Life Technologies, Inc.), penicillin (100 units/ml), and streptomycin (100 /xg/ml). SV40-transformed fibro-

blast cells, GM 637G, (normal), GM 4429E (XPA), and GM 8207B (XPD)were obtained from the Human Genetic Mutant Cell Repository (Camden, NJ).Human topoisomerase I was purified from human placenta as describedpreviously (26). The Scl70 antibody is a human polyclonal antibody fromscleroderma patients that is directed against topoisomerase I; the MPS antibody is a rabbit polyclonal antibody that is directed against the Mr 170,000form of human topoisomerase II (kindly provided by TopoGEN, Inc., Columbus, OH). CPT and etoposide were donated by TopoGEN, Inc. T4 endonucle-

ase V was kindly provided by Dr. Stephen Lloyd (University of Texas MedicalBranch, Galveston, TX). Topoisomerase I assays (relaxation and cleavage)were performed using pHOTl DNA (TopoGEN, Inc.), which contains thehigh-affinity topoisomerase I cleavage site (27).

UVB Irradiation of Cells. Cell monolayers were washed twice with PBS(137 mM NaCl, 2.7 min KC1, 4.3 mM Na2HPO40-7H,O, and 1.4 HIMKH2PO4)

and overlaid with 15 ml of PBS. The cells were then exposed on ice to UVBproduced by two Westinghouse FS40 SunLamps (4.5 J/nr/s), which wasfiltered through the polystyrene flask to remove wavelengths shorter thanapproximately 290 nm (28). Spectroscopic examination of the flask materialwas performed to ensure that the transmission characteristics did not significantly change between flasks. Under these conditions, the DNA damages wereproduced by the 290-310-nm wavelength region of this source (28). In somecases, cells were grown in 100-mm dishes (Falcon), washed with PBS, and

irradiated (plastic lids removed) by four General Electric G15T8 germicidallamps (0.35 J/m~/s). The fluence rates were measured using an International

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DNA REPAIR RECRUITS TOPOISOMERASE I

Light 1700 radiometer. Following irradiation, medium was replaced, and cellswere incubated at 37°C. At various times post-UV treatment, cells were

harvested using the ICT bioassay.ICT Bioassay. At times specified for each experiment, medium was re

moved from the cultures by aspiration, and cells were lysed by the directaddition of 1 ml of 1% sarkosyl (preequilibrated to 37°C).Samples could then

be stored frozen as lysates or processed immediately. Each lysate was overlaidonto a step CsCl gradient containing four different densities. To prepare thestep gradients, a stock solution of CsCI (density = 1.86 g/cc) was diluted withTE buffer [10 mM Tris-HCl (pH 7.5)-1 mM EDTA] to give four solutions of

1.37, 1.50, 1.72. and 1.82 g/ml. The most dense solution (2 mil was placed inan SW41 polyallomer tube first, followed by 2 ml each of the successively lessdense CsCl solutions. Cell lysates were gently layered on top of the gradient.Gradient tubes were then balanced with mineral oil and centrifuged in aBeckman SW41 rotor at 31,000 rpm for 12-24 h at 20°C.Fractions (0.4 ml)

were collected from the bottom of the gradients, and DNA was located (andquantified) using fluorometry. Aliquots from each fraction (100 jxl) werediluted with an equal volume of 25 mM sodium phosphate buffer (pH 6.5) andapplied to Hybond C nitrocellulose membranes (Amersham) using a slot-blot

vacuum manifold.Immunoblotting Analysis. Nitrocellulose membranes were first equili

brated in TBST buffer [10 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1%

Tween 20] for 15 min, followed by incubation in TBST containing 5% nonfatdried milk (Blotto) for 2 h. The filters were washed three times (10 min perwash) in TBST and incubated for 6 h with the appropriate antibody (topoi-

somerase I or topoisomerase II antibody: TopoGEN, Inc.). The antibodies werediluted 1:1000 in TBST prior to use. Filters were washed three times (10 minper wash) in TBST, followed by a 2-h incubation in 10 ml of (I25l]protein A

(ICN) at 0.4 /xCi/ml in TBST. washed three times (10 min per wash) withTBST, and visualized by autoradiography.

Preparation of Extracts for Western Blotting. HeLa cells were washedtwice with PBS and lysed by the addition of 1% sarkosyl. Equal amounts oftotal protein from each harvest were analyzed by SDS-PAGE, followed byelectroblot transfer to nitrocellulose. Blots were probed with Scl70 anti-

topoisomerase I antibody.Quantitation of Topoisomerase I-DNA Covalent Complexes. Topoi

somerase I signals in the DNA peak region of the CsCl step gradients werescanned by densitometry and compared to a standard curve, which wasgenerated using known concentrations of purified topoisomerase I on the sameimmunoblot to determine the absolute amount of topoisomerase I (in ng) ascovalent complexes. Only light exposures of the negatives were scanned toensure linearity of the signals. The DNA concentration in each fraction wascarefully measured by fluorometry (using known DNA concentration standards). The concentration of topoisomerase I-DNA complexes is expressed as a

ratio of topoisomerase (in ng) per /j.g of genomic DNA. Thus, the yield ofcovalent complexes is expressed as the amount of covalently bound topoisomerase per cell because DNA concentration is constant.

Topoisomerase I Activity Assays. Reactions containing 250 ng of negatively supercoiled plasmid DNA (pHOTl) and human topoisomerase I in afinal volume of 20 /¿Iin 1X TGS buffer [10 mM Tris-HCl (pH 8.0), 1 mM

EDTA, 150 mM NaCl. 5% glycerol, 0.1% BSA, and 0.1 mM spermidine] wereincubated for 30 min at 37°C.Reactions were stopped by the addition of 5 ml

of stop buffer (5% sarkosyl, 0.125% bromphenol blue, and 25% glycerol). Anequal volume of chloroform:isoamyl alcohol (24:1) was added, and the solution was vortexed and centrifuged (30 s in a microfuge). The aqueous (blue)layer was directly loaded onto a 1% agarose gel in TAE buffer (40 mMTris-acetate-2 mM EDTA) containing 0.5 mg/ml ethidium bromide. Gels were

run at 5 V/cm for 2 h, destained with distilled water for 30 min, andphotographed. DNA band intensities were analyzed using an image quantification program (ImageQuant; Molecular Dynamics) to quantify the reactionproducts. To detect topoisomerase I-DNA cleavage complexes, reactions were

stopped by the addition of SDS to 1%, followed by 50 mg/ml proteinase K for30 min at 37°C,prior to chloroform/isoamyl alcohol extraction as above, and

agarose gel analysis.Preparation of End-labeled DNA Fragments and UV Irradiation. A 3'

uniquely end-labeled 500-bp fragment was generated from pHOTl DNA byfirst digesting with EcoRI and then filling in the 3'ends (DNA polymerase I

and radioactive nucleotide). followed by a second digestion with Sspl.Uniquely end-labeled fragments were separated from the parental fragment on

preparative polyacrylamide gels, followed by electroelution and ethanol precipitation. UV-irradiated DNA was prepared by exposure to 254-nm UVC light

from a germicida! lamp at a fluence rate of 2 J/nr/s. The samples were kept onice to avoid heating and evaporation. The doses used in the study ranged from0 to 2000 J/m2. The numbers of CPDs produced at each dose were as follows:50 ]/m2. 1 CPD/1600 bp; 100 J/m2, 1 CPD/800 bp; 200 J/m2, 1 CPD/400 bp;400 J/m2. 1 CPD/200 bp; 800 J/m2. 1 CPD/IOO bp; 1200 J/m2, I CPD/70 bp;and 2000 J/m2. 1 CPD/40 bp.

Topoisomerase I Cleavage Assays. The 500-bp EcoRl-Sspl fragment de

scribed above was incubated with purified human topoisomerase I (40 nM) in20-ml reactions containing 10 mM Tris-HCl (pH 8.0). 60 mM NaCl. 3 mMCaCl2, 0.1 M sucrose, 1 mM DTT, 100 mg/ml BSA, and 5% glycerol at 37°C

for 30 min (29). Cleavage reactions were terminated with SDS and EDTA ( 1%and 10 mM final concentrations, respectively), followed by a 10-min incubation at 45°C. Following the addition of NaCl to 0.8 M, the samples were

precipitated with ethanol and digested with 250 mg/ml proteinase K in 0.5%SDS at 37°Cfor 30 min. An equal volume of loading buffer [0.05% brom

phenol blue. 0.05% xylene cyanol, and 5 mM EDTA (pH 8.0) in deionizedformamide] was added, and samples were adjusted with loading buffer tocontain the same number of cpm/ml, heated to 95°Cfor 5 min, and loaded ondenaturing polyacrylamide sequencing gels (8%). The 500-bp EcoRl-Ssp\ 3'

end-labeled fragment was also treated with T4 endonuclease V to determine

the location of CPDs using protocols described previously (30).Quantitation of Pyrimidine Dimers in Vivo. DNA fractions recovered

from CsCl gradients were denatured by the addition of I M NaOH (5 min atroom temperature), neutralized with HC1, and directly applied to a nitrocellulose membrane (Hybond C; Amersham) that was presoaked in I M ammoniumsulfate. Filters were soaked in 5X SSC (0.75 M sodium chloride-0.075 Msodium citrate) for 10 min (room temperature), baked under vacuum (80°Cfor2 h). and placed in 1% gelatin (in PBS) at 37°Covernight. Filters wereincubated at 37°Cfor 6 h with the UV3 antidimer antibody (kindly provided

by Dr. A. Wani, Department of Radiology, Ohio State University, Columbus,OH), diluted 1:5000 in PTNB (PBS containing 0.05% Tween 20, 0.5% normalgoat serum, and 0.5% BSA), washed three times (5 min each) in PBScontaining 0.1% Triton X-KX), and then incubated with [I25l]protein A (4 /iCi

in 10 ml of PTNB) for 2 h at room temperature. After washing (three PBSwashes at 5 min per wash), filters were dried and exposed to film.

RESULTS

Analysis of Topoisomerase-DNA Covalent Complexes in I V15-irradiated Cells. To evaluate the role of topoisomerases in UVB-

induced DPCs and in nucleotide excision repair, HeLa cells weretreated with UVB and then placed back in the incubator and analyzedat various times postirradiation by the ICT bioassay (24). The ICTbioassay is an antibody-based method that detects endogenous topoi-somerase-DNA covalent complex formation in vivo. Cells are lysedby sarkosyl, which denatures proteins and traps topoisomerase-DNAcovalent complexes while dissociating noncovalent protein-DNA

complexes (histones. transcription factors, and so on). Covalent complexes are then separated from free proteins by cesium chloridegradient centrifugation. Because DNA and protein band at very different densities ( 1.7 and 1.3 g/ml, respectively), there is no cross-over

between free protein and free DNA in these gradients. Covalenttopoisomerase-DNA complexes band with the DNA-containing frac

tions. Antibodies specific to topoisomerases are used to probe theDNA-containing fractions on immunoblots to detect topoisomerase-

DNA covalent complexes. Thus, the ICT bioassay can be used todirectly evaluate the action of endogenous topoisomerases ongenomic DNA in response to repair-related activity.

HeLa cells that were not exposed to UV show relatively low levelsof topoisomerase I and topoisomerase II covalent complexes (Fig. 1,A, Lanes 2 and 3, and B, Lanes 1 and 6). This was expected becausethe rate of religation in the catalytic cycle of topoisomerases is muchfaster than the rate of cleavage and formation of the strand-joiningproduct is strongly favored (8-10); therefore, the covalent complex is

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DNA RRPAIR RECRUITS TOPOISOMERASE I

Topo I Control

iOHMCPT

Topo II Control

NoDrug

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Topo I

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Topo II

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Hours Post UVTreatment

topoisomerase I and DNA (31-33). This results in a change in the

cleavage/religation equilibrium that facilitates trapping of the topoisomerase I-DNA cleavage/covalent complexes. Similarly, etoposideis a topoisomerase II poison that stabilizes the 5' covalent complex

formed between topoisomerase II and DNA (8-10). Topoisomerase I-and topoisomerase II-DNA covalent complexes can readily be de

tected in the presence of CPT and etoposide, respectively, demonstrating the utility of the assay (Fig. IA, Lanes 1 and 4). Topoisomerase I-DNA covalent complex formation was also clearly seen in cells

treated with UVB irradiation even in the absence of CPT. The topoisomerase I/UVB response was detected within minutes after exposureto 10 kJ/m2 UV, and complex formation continued to increase after

irradiation (Fig. \B, Lanes 2-5). We refer to this phenomenon as thetopoisomerase I/UV response. In contrast, topoisomerase II-DNA

adducts were not detected at any time following UVB treatment,indicating that covalent complex formation is specific to topoisomerase I and not likely to be due to nonspecific cross-linking of proteinson DNA (Fig. Iß,Lanes 6-10). To examine the possibility of nonspecific DNA-protein cross-linking in more detail, cells were treated

with UVB or CPT and exposed to l MNaCl just prior to sarkosyl lysis.These are conditions that alter the cleavage/religation equilibrium oftopoisomerase I and favor the elution of the enzyme off the DNAsubstrate, thereby significantly reducing the yield of covalent complexes. High salt treatment results in a 87% reduction in CPT-inducedtopoisomerase I-DNA covalent complexes and an 89% reduction in

g 10

Fig. 1. UVB irradiation induces the formation of topoisomerase I-DNA covalentcomplexes. HeLa cells (1 X IO*1cells per treatment) were analas described in "Materials and Methods." A. control experiments with topoisomerase I and

topoisomerase II poisons. For topoisomerase I (Topoisnmerase /). HeLa cells were treatedwith 10 mM CPT (or no drug) for 30 min at 37°C.followed by ICT bioassay analysis with

the Scl70 anti-topoisomerase I antibody. For topoisomerase II (Topoisomerase II}, cells

were treated with 50 mM etoposide (Elup.i or no drug for 30 min. and the blot was probedwith MPS antibody to topoisomerase II (specific to pl70 form). Note that the antibodybinding to fractions at the top of the gradient is nonspecific due to excess aggregates ofcell membranes, debris, free proteins, and so on. The ICT bioassay does not rely on signalsderived from this region of the gradient. B. topoisomerase I and topoisomerase II responseto UVB irradiation. HeLa cells were treated with 10 kJ/irr UVB irradiation. Followingexposure to UVB. plates were immediately placed in the 37"C incubator for

prior to harvest. Cells were analyzed by the ICT bioassay using either anti-topoisomeraseI and anti-topoisomerase II antibodies. The DNA containing fractions from both blots are

marked to illustrate the location of covalent complexes. The DNA peak in Lane 5shifted up the gradient by roughly one fraction; however, this was not seen in otherexperiments and, therefore, reflects slight differences in fractionation between gradients.

difficult to detect in the absence of specific poisons. For example, ina control experiment, nonirradiated HeLa cells were treated with 10mM CPT or 50 mM etoposide for 30 min at 37°C.CPT. a topoisomerase I poison, stabilizes the 3' covalent complex formed between

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Fig. 2. Reversal of UVB-induced topoisomerase I-DNA covalent complexes. HeLacells were treated with UVB at a fluence of 10 kJ/m2 or with 10 mM CPT and thenincubated for 30 min at 37°C.For each treatment, one Petri plate was treated with NaCl

to a final concentration of l Mjust prior to sarkosyl lysis. The other Petri plate was directlylysed with sarkosyl. Covalent complex formation was analyzed by the ICT bioassay asdescribed.

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DNA REPAIR RIZCRUITS TOPOISOMERASE

Fig. 3. Time- and dose-dependenl analysis of UVB-induced topoisomerase I-DNA covalent complexes.A-C, kinetic analysis of UVB-induced topoisomeraseI-DNA covalent complexes. HeLa cells ( 1 X IO6 cells

per treatment) were treated with UVB irradiation at afluence of either 7.5 (Ai, IO (ß).or 20 (O kJ/trr andanalyzed at various times with the 1CT bioassay. Theyield of topoisomerase I-DNA covalent complexes wasquantified as described in "Materials and Methods."

The experiment was performed in duplicate (D and O,duplicate data points). D, dose dependence of topoisomerase I-DNA covalent complexes. HeLa cells weretreated with UVB fluences of 0, 0.3, 1. 3. and 10 kJ/nrfor 5 h followed by ICT bioassay. Data points, averagesof four independent experiments.

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UVB-treated cells (Fig. 2). Thus. UVB-induced topoisomerase I-DNAcovalent complexes appear to behave in a similar manner to CPT-

induced complexes.The kinetics of the topoisomerase I/UV response were analyzed at

different doses of UVB (Fig. 3, A-C). Topoisomerase I-DNA covalentcomplexes could be detected within 12 min post-UVB (1.5-3-fold

increase over no incubation), and the topoisomerase I/UVB responsecontinued to increase up to 5 h posttreatment (3-8-fold over no

incubation). HeLa cells were also treated with UVB fluences over awider range (from 0.3 to 10 kJ/m2) and tested at 5 h postirradiation for

the topoisomerase I/UVB response (Fig. 3D). The results show thatthe topoisomerase I/UV response increases with dose and can bedetected at doses as low as 0.3 kJ/m2.

To evaluate the effects of UVB exposure on topoisomerase Ipolypeptide levels. Western blots were carried out at different timesafter irradiation (Fig. 4). The data reveal that the early topoisomeraseI response (from 0 to 12 min postirradiation) was attended by a slightelevation (estimated to be 20% or less) in stable topoisomerase Ipolypeptide levels (Fig. 4, inset). At later times, when the topoisomerase I/UV response reached a plateau (1.5 and 5 h), we observed anadditional increase (as much as 1.5-2-fold) in topoisomerase Ipolypeptide levels. The 1.5-2-fold increase in topoisomerase I protein

levels, which is reproducible, may be explained by the general effectof UV on gene expression (reviewed in Ref. 34). UVC at much lowerfluence than UVB also causes a clear elevation in topoisomeraseI-genomic DNA complex formation, and the kinetics of UVB and

UVC responses were essentially identical (data not shown). Also notethat the kinetics of UV dimer repair (Fig. 4. open circles) demonstratethat the rapid rise in topoisomerase I-DNA complex formation (0-2 h

postirradiation) is attended by a commensurate decrease in UV lesionsas the excision repair process get underway.

UVB-induced Topoisomerase I-DNA Covalent Complexes inG,-arrested and Exponentially Growing Cells. If topoisomerase-

DNA covalent complexes were formed as a result of DNA repairfollowing UVB exposure, it would be expected that similar yields ofadducts would form in serum-arrested G, cells compared with expo

nentially growing cells. This is based upon the finding that DNArepair levels are similar in cells cultured under these conditions (35).To address this point, topoisomerase I-DNA covalent complexes werecompared in G,-arrested and exponentially growing diploid fibroblastcells. Both G |-arrested and exponentially growing cells displayed a

large increase in topoisomerase I adduci formation following UVBirradiation, with overall levels of complex formation being slightlyhigher in G,-arrested cells (Fig. 5A). The data show that complex

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DNA REPAIR RECRUITS TOPOISOMERASE

2.0 100

Time postirradialion (hours)

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Fig. 4. Topoisomerase 1polypeptide and CPD levels in UVB-treated HeLa cells. HeLacells (1 X 10* cells per treatment) were treated with a DVB fluence of 10 U/m2. and at

indicated times, cells were harvested. Equal amounts of protein from each lysate wereloaded on a 7.5% SDS-PAGE and analyzed by Western blotting techniques using amonospecific anti-topoisomerase I antibody (inset). Covalent complex formation (•)was

measured at the indicated times using the ICT bioassay. Data points, average of threereplicate experiments; bars. SD. CPD formation on genomic DNA (O) was measured onDNA samples recovered from the CsCI gradient fractions in the ICT assay. The peak DNAfractions were pooled, and concentration was determined by fluorometry. Three concentrations of DNA (5. 2.5, and 1 fig of DNA) were processed. The total amount of CPD wasdetermined by Western slot blotting using an antidimer antibody, as described in the"Materials and Methods." Relative levels of CPD formation are expressed as a percentage

of the maximum value (measured immediately after UV exposure) and corrected forbackground CPD levels (antibody signal from cells not exposed to UV).

formation does not correlate with S-phase; in fact, G, cells displayedmarginally higher levels of topoisomerase I-DNA covalent complexes. CPT treatment, on the other hand, induces 2-fold more topoisomerase I-DNA covalent complexes in exponentially growing cellsthan it does in G,-arrested cells (Fig. 55). The results demonstrate thatthe topoisomerase I/UVB response is S phase-independent, as would

be expected for a process that is linked to DNA repair. In contrast,CPT-induced topoisomerase I-DNA covalent complexes are enhancedin exponentially growing cells (compared to G,-arrested cells), whichsupports the notion that CPT-mediated killing is linked to S phase (8).

Effect of UV Photoproducts on Topoisomerase I CatalyticActivity. It has recently been shown that UV photoproducts or DNAmismatches inhibit topoisomerase I activity in vitro (36, 37). Thebasis of this observation is thought to be due to alterations in thecleavage/religation equilibrium of topoisomerase I on UV-damaged

DNA targets, thereby increasing the life expectancy of cleavagecomplexes. Conceivably, the topoisomerase I/UV response could beexplained by an inhibition of the topoisomerase I religation step,leading to an accumulation of the covalent intermediate (similar toconventional topoisomerase I poisons). The effect of UV photoproducts on the ability of human topoisomerase I to relax supercoiledDNA was evaluated. Supercoiled plasmid DNA (pHOTl) was treatedwith increasing UVC doses from 0 to 2000 J/m2 and incubated with

purified human topoisomerase I. Reaction products were quantified bydensitometric scanning of gel data and plotted as a function oftopoisomerase I (Fig. 6). The control experiment shows that CPTclearly inhibits topoisomerase I relaxation activity at all concentrations of drug tested (Fig. 6A). Likewise, UV lesions in irradiated DNAalso inhibit the relaxation activity of topoisomerase I. In the absence

of CPT, topoisomerase I relaxed approximately 35% of the inputDNA substrate, whereas in the presence of CPT, the yield of relaxedDNA was reduced by 3-fold (to 12%). Comparing normal DNA andUV-irradiated DNA substrates (Fig. 65), we detected a 4-fold reduction in topoisomerase I-mediated relaxation with UV-treated DNA.

These results reveal a dose-dependent reduction in topoisomerase I

relaxation activity in the presence of UV lesions that is similar to the

effect seen with CPT.To determine whether the UV dependent inhibition of topoisomer

ase I might be due to cleavage complex stabilization, the yield ofnicked DNA was determined with a normal and UV-damaged DNAsubstrate (Fig. 7). At the highest UV dose, there is a 3-fold increase

in topoisomerase I-DNA covalent complexes (55% open circular

DNA), compared to untreated DNA (18%). This is consistent with the3-fold decrease in topoisomerase I relaxation activity. Moreover,UV-stabilized topoisomerase I cleavages in vitro are readily reversible

(data not shown), which is consistent with the in vivo data (Fig. 2; seeabove). Thus, UV lesions alter the topoisomerase I cleavage/religationequilibrium, resulting in stabilized enzyme-DNA cleavage complexes.

Stabilized cleavages in and around UV lesions might be explained byan accelerated initial incision step or a less robust resealing step (or acombination of these two steps).

0.0

Time Posttreatment (hours)

Fig. 5. Topoisomerase I complex formation in G,-arrested and exponentially growing

cells. GM4390 cells (a diploid human fibroblast cell line) were arrested in G, in mediumcontaining 0.5% fetal bovine serum. G, arrest was verified by (3H]thymidine pulse

labeling. An exponentially growing cell population was prepared by seeding cells in 10%fetal bovine serum 36-48 h prior to treatment. The cultures were treated with a UVBfluence of 10 kJ/m2 and analyzed at various times post-UVB treatment for topoisomeraseI-DNA covalent complexes using the ICT bioassay. Inset, exponentially growing and

Grarrested GM4390 cells that were not UVB irradiated were treated with 10 fíMCPT for30 min at 37°C.followed by analysis by the ICT bioassay (see Fig. \A). Data points,

average of three replicate experiments; bars. SD.

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UNA KUI'AIR RWRIÕTS TOPOISOMI.K ASI

10 20 30 40 50CPT Concentration (U.M)

60

500 1000 1500UV Dose(J/m2)

2000

Fig. 6. Topoisomerase I relaxation assays in the presence of camplothecin (A) and UVlesions (ß).Purified human topoisomerase I (5 nM) was incubated with supercoiledpHOTl for 30 min at 37°C. and reaction products were analyzed by agarose gel

electrophoresis. Formation of relaxed DNA measured was determined as described in"Materials and Methods." All reactions were performed in quadruplicate.

Nucleotide Localization of Topoisomerase I-mediated DNABreaks Stimulated by UV Photoproducts. Topoisomerase I-medi

ated cleavages in DNA are determined by weak sequence specificity(38, 39) as well as DNA conformational features (i.e., bends orcurves; Refs. 40-42). UV lesions introduce considerable deformationin the DNA helix that could stabilize topoisomerase I-DNA covalent

complexes (see above). Therefore, the influence of UV photoproductson topoisomerase I cleavage was characterized at the single nucleotidelevel. A 500-bp 3' end-labeled probe was incubated with topoisomer

ase I, and reaction products were analyzed on sequencing gels. Topoisomerase I cleavage sites in UV treated DNA were compared withcleavages in the presence of CPT. Additionally, to locate cleavagesrelative to CPD sites, the DNA was digested with T4 endonuclease V.Five prominent topoisomerase I cleavage sites seen in the absence ofCPT or UV (Fig. 8, Lane 4) are marked TI(1)-TI(5); however, these

bands are faint, consistent with the rapid religation of topoisomeraseI during its normal catalytic cycle (see also Fig. 7). In the presence ofCPT, topoisomerase I cleavages at these sites and several others areclearly enhanced (Lanes 9 and 10). UV treatment results in a dose-

dependent increase in the intensity of three of the cleavage bands,labeled TI(1), TI(3), and TI(5). Analysis of the relative distribution of

UV-induced CPDs reveals that these three topoisomerase I cleavage

sites occur in close proximity to CPD clusters. Cleavage at TI(2)appears to be unaffected by UV lesions. Note that there are nocontiguous CPD sites near TI(2). Although TI(4) is very close to aCPD site, it was unaffected by the contiguous CPD lesion; however,a UV-dependent topoisomerase I site. UV( 1), appears to be essentiallyon top of this CPD lesion. Another UV-dependent site, UV(2), is

directly on top of a CPD lesion [and there are others above the TI( 1)site in Lanes 6-8]. These UV-dependent sites share the common

feature of being in the immediate vicinity of a CPD lesion.Topoisomerase I-DNA Complex Formation in Repair-deficient

Systems. Topoisomerase I action at UV-damaged and abasic DNA

lesions might promote formation of terminal cleavage complexes(Refs. 36 and 37 and this study). This alone might explain our findingsthat, following UVB irradiation, endogenous topoisomerase I-genome

covalent complexes are elevated in vivo. To further examine this,repair-deficient cell lines were tested using the ICT bioassay. Diploid

fibroblasts from normal donors or from patients exhibiting XPA andXPD were examined for the topoisomerase I/UVB response (Fig. 9).These repair-deficient lines are very sensitive to DNA damage andsustain as many UV lesions as do wild-type cells for each equivalent

dose; however, given that the excision repair pathway is compromisedin the mutants, lifetime expectancy for UV damages is protracted(data not shown). If dead-end, terminal complexes explain the topoi

somerase I/UV response (see Fig. 1), then one might expect to see aneven greater degree of complex formation in the mutant cell line. TheXPA cells show a reproducibly lower topoisomerase I/UV response(compared to wild-type cells, although kinetics are still similar tothose of wild-type cells; Fig. 9 and Table 1). The XPD cells are

strongly deficient in their ability to recruit topoisomerase I into

100

80 -

60 -

U

iO 40 -

20 -i.

OI/m1 «OI/m' 800I/m' 1200I/m' 2000I/m1Topol - T "- + ~- Õ - + - -f

Open Circulai DNA

Supercoiled DNA

Relaxed DNA

_J I + Topo I

- TopoI

500 1000 1500UVDose(J/m2)

2000 2500

Fig. 7. Dose dependence of topoisomerase I-DNA covalent complex formation in vitrtt.

DNA relaxation assays were performed using unirrudiated or UV irradiated pHOTl DNAand 20 nM of purified human topoisomerase I; cleavage complexes were measured asdescribed in "Materials and Methods." The gels were quantified by densitometry asdescribed in "Materials and Methods." Insel, ethidium bromide agarose gel analysis of

products. Reactions were performed in triplicate.

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DNA REPAIR RECRUITS TOPOISOMERASE I

e

Io

UV DNA+ EndoV

l 2 3

UV treated DNA/Topo I

45678

Non UV DNA+TopoI+ CPT

9 10

1-TI(l)

are intermediates formed during the catalytic cycle of topoisomeraseI and reflect total topoisomerase I action on genomic DNA. Undernormal conditions, covalent topoisomerase-DNA complexes cannot

be arrested by sarkosyl lysis because the lifetime of the cleavagecomplex is very short (or the religation step is rapid; Refs. 23 and 32):therefore, little if any topoisomerase is detected in the DNA peakusing antibodies with ICT bioassay (24, 25). Topoisomerase I poisonsprolong the half-life of topoisomerase I-DNA covalent complexesand, thus, increase the yield of complexes (29, 31-33). In contrast,

cells treated with UVB exhibit an enhancement in topoisomeraseI-DNA covalent complex formation without the need for CPT tostabilize the complexes. When topoisomerase I-DNA covalent com

plexes are trapped on the DNA as part of the normal catalytic cycle of

'UVU)

Fig. 8. Sequencing lopoisomerase I cleavage sites in UV-trealed and untreated DNA.The 3' end-labeled EcoR\-Sspl probe (500 bpl was UV irradiated with various doses andincubated with 40 nM of purified human topoisomerase I for 30 min at 37°C.and the

reactions were terminated by adding SDS. Reaction products were analyzed on a 8%polyacrylamide sequencing gel. Lane I. untreated probe alone: Lanes 2 and 3, endonu-clease V and DNA treated at 40(1 and 2000 J/nr. respectively; Lanes 4-fi. topoisomeraseI and DNA treated at 0. 4(X), 800. 1200 and 2(XK)J/nr, respectively; and Lanes 9 and 10.non-UV-irradialed DNA with topoisomerase I plus 10 and 50 mM CPT. respectively.

covalent complexes following UV exposure (Fig. 9). Both mutant celllines contain essentially identical amounts of topoisomerase I, bothbefore and after UV treatment, compared to normal cells (based onWestern blots; data not shown); thus, our results are not due todifferences in protein levels. More importantly, we found that theXPA and XPD CPT response was similar or even somewhat largerthan that of wild-type cells (Fig. 9, inset, shows topoisomerase I

covalent complexes in ng protein//ig DNA; Table 1 expresses the dataas ratios before and after CPT or UV). From this result, we concludethat the total amount of cellular topoisomerase I available for deployment onto the genome is not substantially different between thesethree cell lines. In contrast, XPA cells and. to a greater extent, XPDmutants were quite strongly depressed in terms of topoisomerase Irecruitment at 5 h after UVB treatment (Table 1).

DISCUSSION

In this work, we report that treating human cells with UVB irradiation stimulates endogenous topoisomerase I-DNA covalent com

plexes in vivo during the period of DNA repair and recovery. [Identical results are obtained with equivalent doses of UVC irradiation andother agents that damage DNA (data not shown).] These complexes

0.8-

- UV(2) ff

12345Hours After UV Treatment

Fig. 9. Topoisomerase I/UV and CPT response in wild-type and repair-deficient celllines. Cell lines derived from XPA and XPD were treated with UVB (10 kj/nr) andanalyzed at various times posttreatment for the formation of topoisomerase I-DNAcovalent complexes using the ICT bioassay. Normal human diploid fibroblasts were usedas a control. Dala paints, average of four experiments; bars. SD. Inset, CPT response foreach cell line, as determined by the ICT bioassay. The data (average of four differentexperiments) in this table correspond to ng of topoisomerase I complex per ¿igof cellDNA.

Table 1. Topoisomerase 1-DNA complex formation in normal and XPA-XPD-derivedcells"

Cell line +CPT:-CPT ratio + UV:-UV ratio

Wild typeXPAXPD

13.515.418.8

4.74.91.45

" Wild-type or repair-deficient cell lines were treated with UVB or CPT (as described

in legend to Fig. 9) and analyzed for endogenous topoisomerase I-DNA covalent complexes using the ICT bioassay. Covalent complex formation was quantified as ng oftopoisomerase I/fig DNA. The data are expressed as an index ratio (no drug to drugtreatment or no UVB to UVB treatment, at the 5-h time point; see Fig. 9) to reflect therelative increase in topoisomerase I covalenl complexes as a result of either UVB or CPTexposure (as described in "Materials and Methods"). The data represent the average of

four independent experiments.

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DNA REPAIR RKCRUITS TOPOISOMliRASli I

topoisomerase I (detergents are used to denature the intermediates),complexes are readily reversed by either drug removal or extraction with high salt. Treatment with high salt reverses the UV-induced complexes to the same extent, as seen with CRT-dependent complexes (Fig. 2). Nonspecific DNA-protein UV cross-linking should not be salt labile. Additionally, the topoisomeraseI/UVB response was found to be aborted in a repair-deficient cellline (Fig. 9). If the topoisomerase I/UVB response was due solelyto UV-induced cross-linking or some other stable complexes unrelated to topoisomerase I activity, it is unlikely that the levels ofcomplexes following UV irradiation would differ between repair-proficient and -deficient cell lines. In addition, other DNA-bindingproteins (topoisomerase II) did not become trapped on DNA afterUV irradiation; thus, the effect is specific to topoisomerase I. Amore likely interpretation is that the UV irradiation results inalterations of endogenous topoisomerase I catalytic activity ongenomic DNA. It also seems rather unlikely that our results can beexplained by widespread cell death after UV irradiation. First,covalent complex formation displays a linear response with doseand can be detected at UVB doses as low as 0.3 kJ/m2, which result

in relatively little cell killing (as determined by flow cytometry;data not shown). Second, the topoisomerase I/UVB response wasdetected within minutes after UVB exposure, well before cytotox-icity and/or apoptosis. Third, we noted that, 5 h after UVB exposure, there was a 1.5-2-fold increase in topoisomerase I polypep-tide levels. Thus, the cells appeared to be competent in new proteinsynthesis after UV irradiation. Finally, given that repair-deficientcells are defective in the UVB-dependent topoisomerase I response, cell death alone could not explain our findings. It is worthnoting that the UV-deficient cell lines are hypersensitive to UV;thus, one might expect even greater cytotoxicity and cellulardemise with the mutants. In other words, one can dissociate thetopoisomerase I/UVB response from events related to cell death.

Our in vitro and in vivo results with topoisomerase I are mutually consistent with those reported by Lanza et al. (36), whoshowed that CPD and (6-4) photoproducts interfere with the propercatalytic activity of topoisomerase I, leading to a stimulation incleavage complexes. We estimate that DNA relaxation activity wasreduced by approximately 30% when ~10 lesions were introducedper 400 bp of DNA. Decreased relaxation activity correlates withan increase in topoisomerase I-DNA cleavage complexes, whichmay be due to a less robust religation step. In addition, topoisomerase I-DNA covalent complex formation in vitro increases linearlywith UV dose. A similar result is seen with CPT and normalB-DNA. Thus, the in vivo UV-topoisomerase I response may (atleast partially) be explained by the formation of stabilized topoisomerase I-DNA cleavage complexes. For example, localization oftopoisomerase I cleavages at the nucleotide level revealed that UVlesions stimulate cleavage sites in close proximity to CPD clusters.The presence of these CPDs may alter the local DNA conformationat the break sites, causing misalignment of the two ends of thebreak site and leading to a stabilized broken intermediate (see alsoRef. 36).

Altered cleavage/religation equilibria at or near UV lesionshowever, cannot completely explain the in vivo response of topoisomerase I to UVB. Other response parameters clearly impact onthe topoisomerase I/UVB response identified in our study. Forexample, association with other proteins, notably p53, could elevate the endogenous activity of topoisomerase I and enhance itscatalytic deployment to sites of repair (43). When coupled withsome form of repair site recruitment, general activation of topoisomerase I (by p53, for example), could explain our in vivofindings. It is relevant to note that repair-deficient XPA and XPD

cells should have sustained the same amount of photodamage asdid the wild-type fibroblasts. yet the topoisomerase I response isclearly aborted in the mutants. These data suggest that topoisomerase I is actively involved in the repair process at or near the lesionsite and may additionally interact or act in concert with the XPDhelicase. Although there is no known role in vivo for topoisomerase I in DNA repair, the enzyme has been implicated indirectly inDNA repair processes (17, 19, 20, 44). Recent evidence for atopoisomerase I-p53 connection strongly supports this notion because p53 may itself play an important role in DNA repair [seeGobert et al. (Réf.45 and references therein)]. There is additionalstrong evidence that several factors involved in transcription alsoplay a role in repair (45-47). Topoisomerase I localizes to sites ofactive transcription (11-14), and recent evidence suggests thattopoisomerase I is actively recruited through its interaction withTFIID (12, 14). It is not clear at this juncture whether protein-protein interactions and/or altered DNA architecture (bending/supercoiling) might drive topoisomerase I recruitment into tran-scriptionally active chromatin. A parallel scenario may exist inDNA repair-active chromatin. In UV-irradiated cells, lesions arethought to be detected by repair complexes scanning the DNA forconformational abnormalities due to CPDs and (6-4) photoproducts (49). It is possible that topoisomerase I senses these helicaldistortions because it also responds to DNA mismatches and abasicsites in vitro (37, 50). Nucleotide excision repair involves damagerecognition, incision of the damaged strand on opposite sides of thelesion, excision of the lesion containing oligonucleotide, synthesisof new DNA using the undamaged strand as template, and ligation(for reviews see Refs. 2, 51, and 52). Topoisomerase I may berequired simply to adjust DNA topology at any or all of these steps.For example, helix unwinding in the vicinity of the lesion mayintroduce localized supercoiling at the repair site, similar to thatproposed for transcription coupled DNA supercoiling (53). Topoisomerase I has been shown to have a greater affinity for super-coiled DNA (40, 54, 55), and through this mechanism, it could beactively recruited. Stimulation of topoisomerase I catalytic activityby p53 association provides a means to rapidly mobilize preexisting caches of enzyme for recruitment (43). In support of thisnotion, we recently found that the topoisomerase I/UV response isnot affected by cycloheximide.4 Future studies will need focus on

the protein-protein interactions that are pivotal to topoisomerase Ientry into the DNA repair process.

ACKNOWLEDGMENTS

We thank M. Thakar for expert technical assistance and J. Spitzner for

helpful comments.

Note Added in Proof

A recent report by Vichi et al. (56) describes a connection between TBP/TFIID in response to DNA damage. These authors propose that core elementsof the RNA polymerase II transcription machinery are lured inlo UV-damaged

sites in vivo. Their model, which is wholly consistent with our findings,suggests a plausible mechanism in vivo by which topoisomerase I (as amember of the transcription complex) could he recruited into DNA damagesites. Our data would further suggest that the response demonstrated by Vichiet al. (56) happens within minutes after DNA damage is inflicted.

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1998;58:976-984. Cancer Res   Deepa Subramanian, Barry S. Rosenstein and Mark T. Muller  DNA Repair

: Possible Relationship within VivoI-DNA Complex Formation Ultraviolet-induced DNA Damage Stimulates Topoisomerase

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