.J. Mol. BioE. (1988) 202, 333-342

Sequence-dependent Effect of Camptothecin on Human Topoisomerase I DNA Cleavage?

Eigil Kjeldsen ‘y2, Steen Mollerup’, Bo Thornsen’ Bjarne Juul Bonven l, Lars Bolund2 and Ole Westergaard’z

1 Department of Molecular Biology and Plant Physiology University of Aarhus

DK-8000 Aarhus C. Denmark

2Department of Human Genetics University of Aarhus

The Bartholin Building DK-8000 Aarhus C, Denmark

(Received 3 November 1987, and in revised form 2 February 1988)

We have studied the effect of the antitumor drug, camptothecin, on the interaction of human topoisomerase I with DNA at the sequence level. At a low molar ratio of enzyme to DNA, cleavage is prominent and unique, located at a previously described hexadecameric recognition sequence, while a. number of strong additional cleavage sites appear in the presence of the drug. Camptothecin stimulates cleavage at the recognition sequence less than twofold, whereas cleavage at the additional sites is stimulated up to 200-fold. Camptothecin greatly enhances the stability of the cleavable complexes formed at the additional sites, whereas the complex formed at the hexadecameric sequence is only marginally affected. Cleavage was eliminated at certain sites in the presence of camptothecin. Taken together these observations demonstrate that at least three types of potential eukaryotic topoisomerase I cleavage sites can be distinguished by the use of camptothecin. Comparison of the sequences at the additional cleavage sites in the presence of camptothecin reveals that the most frequently cleaved dinucleotide is TG with no consensus for the flanking nucleotides.

1. Introduction

The topological state of DNA greatly affects its functional properties and plays an important role in processes such as replication, recombination and transcription (Gellert, 1981; Wang, 1985). Among the proteins that control the topology of the eukaryotir genome are type I and type II DNA topoisomerases (Gellert, 1981; Voxberg, 1985; Wang, 1985; Maxwell & Gellert, 1986). The discovery that eukaryotic topoisomerases are the cellular targets for certain antitumor drugs (Nelson et al., 1984; Ross et al., 1984; Tewey et al., 1984; Hsiang et al., 1985) has led to the introduction of these drugs for localization of topoisomerases in chromatin (Yang et al., 1985; Udvardy et al., 1986; Gilmour & Elgin, 1986; Stewart & Schutz, 1987)

t This article is dedicated to Arthur Kornberg on the orcasion of his 70th birthday by a former post-doctoral fellow (O.W.) from his laboratory.

1 Author for correspondence.

and for probing biological functions of these enzymes (Snapka, 1986; Nelson et al., 1986; Chow & Ross, 1987; Andoh et al., 1987; Richter et aZ., 1987). The antitumor drug, camptothecin, inhibits the enzymatic activity of eukaryotic topoisomerase I in

vitro (Hsiang et aZ., 1985) and has severe metabolic effects on mammalian cells, including genome fragmentation and inhibition of RNA and DNA synthesis (Horwitz & Horwitz, 1971; Spataro & Kessel, 1972; Abelson & Penman, 1972). These pleiotropic effects were shown to be mediated via a single cellular target, recently identified as topoisomerase I. Thus, a mutant human lympho- blastoid cell line resistant to camptothecin escapes the metabolic effects, by producing a topoisomerase I that in vitro is shown to be resistant to the drug (Andoh et al., 1987).

Topoisomerase I catalyzes changes in the topological state of duplex DNA by performing single-strand breakage-resealing cycles. During the enzymatic action the enzyme transiently becomes covalently linked to the nicked DNA at the 3’ end.

(XM2-&836/X8/140333- 10 $o3.oo/o 333

0 1988 Academic Press Limited

334 E. K.jeldsen et al.

The transient complexes can be irreversibly trapped by treatment with strong protein denaturants such as sodium dodecyl sulfate thereby capturing the nicked intermediate of the catalytic cycle (Champoux, 1977). We will refer to these inter- mediates as cleavable complexes. It has been reported that camptothecin increases the stability of these cleavable complexes, suggesting that camptothecin inhibits topoisomerase-T-catalyzed IlKA relaxation by blocking the religation half- reaction of the normal catalytic cycle (Hsiang et al., 1985). In the present paper, we demonstrate that camptothecin has a differential sequence-dependent effect on human topoisomerase T DNA cleavage. The results may have relevance for the interpre- taGon of data on the localization of topoisomerase I in chromatin based on the use of camptothecin.

2. Materials and Methods

(a) General Camptothecin was kindly provided by the National

Cancer Institute (lactone form, no. 94600). Stock solutions of camptothecin, 10 mM in 100% dimethyl sulfoxide (DMSOP), were stored in samples at -20°C. DMSO was from Merck. Radioactive nucleotides were obtained from ICK. Klenow large fragment of Escherichia coli DKA polymerase I and restriction endonucleases were obtained from New England Biolabs. Polyclonal rabbit IgG directed against calf thymus DNA topoisomerase I was a kind gift from Dr H.-P. Vosberg, Heidelberg. The cell line Daudi (City of Hope) is a tetraploid derivative of the diploid cell line Daudi isolated from a male with Burkitt’s lymphoma (Junker. 1982).

(b) Cell culture and isolation of topoisomerase I

Daudi cells were grown in RPM11640 supplemented with 10% fetal calf serum and used for the purification of DNA topoisomerase I. DNA topoisomerase I was purified to homogeneity by a modification (Thomsen et al., 1987) of the method of Ishii et al. (1983). One unit (u) of topoisomerase I is defined as the activity giving 50% relaxation of 1 pg supercoiled pBR322 in 30 min at 30°C.

(c) Purification and labeling of restriction fragments

The plasmid pNC1 was constructed by insertion of a synthetic oligonucleotide containing the Tetrahymena topoisomerase I recognition sequence into the EcoRV- &&I sites of pBR322 (for details, see Thomsen et al., 1987). ppu’C1 was digested with the restriction endo- nucleases Hind111 and PwuII, according to the suppliers instructions. The fragments were separated by agarose gel electrophoresis and the relevant fragments were isolated by electrophoretic transfer to DEAE membrane (S&S EA45, Schleicher und Schuell) and recovered according to the supplier’s instructions. The isolated fragments were labeled at recessed 3’ ends using [a- ZP]dATP (3000 Ci per mmol), unlabeled deoxyribo- nucleotide triphosphates and the large Klenow fragment of E. coli DNA polymerase I (Maniatis et al., 1982).

t Abbreviations used: DMSO, dimethyl sulfoxide; bp, base-pairs; kb, lo3 base-pairs.

(d) Gel electrophoresis

Maxam-Gilbert sequencing reactions (Maxam & Gilbert, 1980) and topoisomerase I cleavage reactions were analyzed on denaturing polyacrylamide gel (So/;, (w/v) acrylamide, 0.3% (w/v) N, N-methylene- bisacrylamide, 8 M-urea) run for 2 to 6 h in 89 mM-EDTA (pH 8.3). Protein samples were analyzed on SDS/polyacrylamide gels (15 o/o acrylamide, 0.5 “/b N,N-methylene-bisacrylamide, 0.1 y0 SDS), run overnight in 10 mrv-Tris-base, 200 mllr-glycine, 0.1 o/o SDS (pH 8.3), according to the method of Laemmli (1970), followed by silver-staining (Bio-Rad silver staining kit) or Western blotting (Bio-Rad Western blotting kit).

(e) Reaction conditions

Unless stated otherwise, 3-end-labeled restriction fragments (3 fmol) were incubated with 0 to 80 units of topoisomerase I in 30 ~1 Tris . HCl (pH 8.0), 60 mM-Pu’aCl, 3 miv-CaCl,, 0.1 M-sucrose, 1 min-dithiothreitol. 100 pg bovine serum albumin/ml, 0 to 125 PM-camptothecin, 79/o DMSO and 50/: glycerol for 30 min at 30°C. Induction of cleavage was accomplished by addition of SDS and EDTA to final concentrations of 124, and 10 mM, respectively, followed by incubation for 10 min at 45°C. Finally. lVaC1 was added to 0.8 M. In control samples KaCl and EDTA were added prior to SDS. The samples were then precipitated with ethanol and digested with proteinase K (250 pg/ml in 0.5% SDS, 30 min at 37°C) before addition of 1 vol. of deionized formamide, 0.05% bromphenol blue, 0.03% xylene cyanol. 5 mM-EDTA (pH 8.5). Subsequently, the samples were heated to 90°C for 5 min before loading on denaturing polyacrylamide gels.

(f) Autoradiography

Autoradiography was performed at - 70°C with Kodak XAR-5 film and a DuPont Lightning-Plus intensifying screen.

(g) Quantitation of cleavage

The relative frequency of cleavage was determined by densitometric scanning of the autoradiograms using a Shimadzu dual wavelength thin-layer ChromatoScanner, model CS 903. The frequency of cleavage at a given site was determined by normalization of the absorbancy in the band generated by cleavage at this site to the total absorbancy in the lane.

3. Results

(a) A hexadecameric sequence functions as a highly preferred recognition sequence for

human topoisomerase I

The influence of camptothecin on the cleavage properties of human topoisomerase I was investigated at the nucleotide sequence level in vitro. The enzyme was purified from a Burkitt’s lymphoma cell line to a specific enzyme activity of 10’ units/mg and Figure l(a) shows a silver-stained polyacrylamide gel of the final enzyme preparation. One major band is observed with a M, of 97 x 103, corresponding to the values reported for other mammalian topoisomerases (Gellert, 1981; Vosberg,

Sequence-dependent Effect of Camptothecin in Vitro 335

(a) (b)

Figure 1. Immunoblotting of DNA topoisomerase I. (a) SDS/polyacrylamide gel of purified human topoisomerase I. A 4 fig sample of the purified enzyme was applied to the gel and subsequently stained with silver. At the left, the positions of molecular weight markers are indicated ( x IO-‘). (b) Western blot from a gel like the one in (a), stained with polyclonal rabbit IgG antibodies directled against calf thymus topoisomerase I.

1985). Western blot analysis of gels with polyclonal antibodies directed against calf thymus topoiso- merase I identifies the band as topoisomerase 1 (Fig. l(b)).

A hexadecameric recognition motif has been found to serve as a strong recognition sequence for several eukaryotic type I DNA topoisomerases (Bonven et al., 1985; Christiansen et al., 1987; Busk et al., 1987). To investigate whether human topoisomerase I would recognize this sequence on a foreign DNA background, a plasmid (designated pNC1) was constructed by insertion of a 27 bp synthetic oligonucleotide containing the hexadecameric recognition sequence AGACTTAGA- AAAATTT into the EcoRV and SphI sites of pBR322 (for details, see Thomsen et al., 1987). A 1.6 kb H&dIII-PvuII restriction fragment, including the hexadecameric sequence, was isolated

Hind III

Figure 2. Determination of topoisomerase I cleavage site. The isolated 3’-end-labeled HindIII-PvuII restriction fragment from pNC1 was subjected to Maxam-Gilbert sequencing reactions: lane 1, A > C; lane 2, G+ A reaction; lane 3, C > T. Cleavage reaction with the 3’-end-labeled HindIII-PwuII restriction fragment: 3 fmol of fragment was incubated with 25 units human topoisomerase I for 30 min at 3O”C, the reaction was terminated with SDS and processed for sequencing gel analysis as described in Materials and Methods (lane 4). At the right is shown a schematic representation of the 3’-end-labeled HindIII-PvuII fragment, showing the orientation and the hexadecameric sequence. The location of the topoisomerase I cleavage site is indicated by a broken line with an arrow. The cross denotes the position of the 32P label.

from the plasmid, 3’-end-labeled at the HindI site and used as substrate for the purified human topoisomerase I. Cleavage was studied by incubation of the enzyme with t’he 3’.end-labeled

336 E. Kjeldsen et al

restriction fragment at an enzyme to DNA molar specific T residue in the recognition sequence as ratio of approximately 5, after which the depicted in Figure 2. The location of the cleavage transiently nicked enzyme-DNA intermediate was site is consistent with previous observations using trapped by addition of SDS. The position of topoisomerase I from other eukaryotic sources and cleavage was determined by denaturing rDNA as substrate (Christian&n et al., 1987). polyacrylamide gel electrophoresis of the Densitometry of the autoradiogram reveals that deproteinized DNA fragment in parallel with the approximately 25% of the fragments are cleaved at 3’-end-labeled 1.6 kb restriction fragment subjected the recognition sequence. Minor cleavages displayed to Maxam-Gilbert sequencing reactions. in the upper part of the gel account for less than Topoisomerase I generates one major band with the 5%) of the total cleavage. Taken together it length of 171 bases by cleavage of the HindITT- demonstrates that the hexadecameric sequence PvuII restriction fragment. Thus, cleavage is functions as a high-affinity recognition sequence sequence-specific and occurs immediately 3’ to a independent of flanking sequences.

I 2 3 4 5 6 7 6 9 IO II 12

Figure 3. Topoisomerase-I-induced cleavage of DKA in the presence of camptothecin. A 3 fmol sample of 3’-end- labeled 1.6 kb HindIII-PvuII fragment from ppu’C1 was incubated with 30 units topoisomerase I and different amounts of camptothecin (CPT) for 30 min at 30°C with appropriate controls. Lane 1,O u topoisomerase I, 0 PM-CPT; lane 2,O u topoisomerase I, 125 PM-CPT; lanes 3 to 12, 30 u topoisomerase I, different concentrations of CPT; lane 3, 0 PM-CPT;

lane 4, 0.5 PM-CPT; lane 5, 5 PM-CPT; lane 6, 25 PM-CPT; lane 7, 125 ,UM-WT. The reactions in lanes 1 to 7 were

terminated with SDS. Lanes 8 to 12 correspond to lanes 3 to 7, except that reactions were subjected to 0.8 M-N&I for 10 min at 45°C prior to termination with SDS. The arrow indicates topoisomerase I cleavage at the hexadecameric sequence.

Sequence-dependent Effect of Camptothecin in Vitro 337

(b) Camptothecin alters the cleavage pattern

Recently, camptothecin has been used to localize eukaryotic topoisomerase I cleavage sites in vitro (Yang et al., 1985; Gilmour & Elgin, 1986), as the drug is thought to stabilize the topoisomerase I-DNA transient intermediates formed during enzyme action (Hsiang et al., 1985). Having established a procedure for analyzing the inter-

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b-

action of human topoisomerase I with specific DNA sequences, we examined the influence of camptothecin on this interaction. For this purpose cleavage of the 1.6 kb ElindIII-PvuII restriction fragment of pNC1 was done in the presence of increasing amounts of camptothecin (Fig. 3, lanes 3 to 7). As expected, cleavage in the absence of camptothecin is confined to the hexadecameric recognition sequence (Fig. 3, lane 3), while several

123456769

(b 1

Figure 4. Determination of potential cleavage sites in the presence and absence of camptothecin. Cleavage reactions containing 3 fmol of 3’-end-labeled HindIII-PvuII restriction fragment of pNC1 and increasing amounts of topoisomerase I were incubated in the absence and presence of camptothecin, and terminated with SDS after 30 min at 30°C. (a) The amounts of topoisomerase I were: lane 1, 0 u; lane 2, 1 u; lane 3, 25 u; lane 4, 5 u; lane 5, 15 u; lane 6, 20 u: lane 7, 30 u; lane 8, 40 u; lane 9, 80 u. (b) As in (a), except that the reaction mixtures contained ~OPM- camptothecin. The lower case letters and the bars indicate the positions of cleavages by topoisomerase I starting from the HindIII-labeled 3’ end. Positions a, 171 bases (at the hexadecameric sequence); b, 187 bases; c, 208 bases; d, 222 bases; e, 243 bases; and f, 256 bases.

338 E. Kjeldsen et al.

additional cleavage sites are observed in the presence of even low concentrations of camptothecin (0*5pM; lanes 3 and 4). All of the cleavages had protein at the 3’ end (data not shown). No further change in the cleavage pattern resulted from increasing concentrations of camptothecin (up to 125pM; lanes 5 to 7). The frequency of topoisomerase I DNA cleavage at the hexadecameric sequence is only slightly stimulated by camptothecin. Thus, by densitometric scanning (see Materials and Methods) the degree of stimulation is determined to be less than twofold. In agreement with previous results (Thomsen et al., 1987) the dissociation constant, K,, at the hexadecameric sequence was determined to be approximately lo-” M and almost unaffected by camptothecin (data not shown). Control experiments, in which topoisomerase I was omitted. excluded the possibility that the cleavages are caused by the drug itself (Fig. 3, lanes 1 and 2). Incubation in the presence of 0.8 M-NaCl at 45°C for ten minutes prior to addition of SDS abolished cleavage even when camptothecin was present, demonstrating that the effect of camptothecin on the topoisomerase I-DNA complexes is reversible (Fig. 3, lanes 8 to 12).

(c) Sequence-dependent effect of camptothecin on human topoisomerase I DNA cleavage

The question arises as to whether the topoisomerase I cleavage that was observed at the additional sites can also be seen in the absence of camptothecin. Figure 4(a) demonstrates that an increased molar ratio of enzyme to DNA results in additional cleavages outside the recognition sequence (compare lanes 2 to 6 with lanes 7 to 9). Several of the bands in the cleavage pattern generated under these conditions correspond to those induced by camptothecin (compare Fig. 4(a) and (b)). The relative band intensities vary in the presence and absence of the drug. Thus, the effect exerted by camptothecin on topoisomerase- T-mediated cleavage is clearly sequence-dependent. For comparison, we have tentatively classified the cleavage sites into three categories according to the response elicited by camptothecin. The first class, designated class A, is represented by cleavage at t,he recognition sequence and one minor site (Fig. 4, positions a and d, respectively). Cleavage at these sites is only slightly stimulated by camptothecin. The second class, designated class R, is represented by cleavage at positions b, c and f in Figure 4. The sites are characterized by a considerably higher degree of stimulation of cleavage by camptothecin. As a representative of this class the cleavage site at position b was examined further, since it is minimally affected by the masking effect caused by cleavage at positions more proximal to the labeled 3’ end of the fragment. After densitometry it can be estimated that camptothecin stimulates this site more than 200-fold. The third class, designated class C, is represented by cleavage at position e in

Figure 4(a) (lanes 8 to 9). The sites are characterized by the disappearance of cleavage when camptothecin is present. After very strong exposures of the autoradiogram it is not possible to detect any cleavage in the presence of the drug (data not shown). Since cleavage at position f (Fig. 4(a) and (b)), located distal to position e relative to the label, is stimulated by the addition of camptothecin, it is technically possible to detect cleavage at site e, if present.

(d) CaZ’ and camptothecin stimulate cleavage by , different mechanisms

Previously we have observed that the DNA cleavage at th& hexadecameric recognition sequence by Tetrahytienu topoisomerase 1 is greatly enhanced by the presence of divalent cations (Thomsen et’ al., 1987). Therefore, the cleavage properties of the human enzyme were analyzed in the presence and absence of divalent cations. The result of such an experiment is shown in Figure 5.

3

Figure 5. Effect of divalent cations on DNA cleavage. Cleavage reactions containing 3 fmol of 3’-end-labeled HindIII-PvuII restriction fragment of ppu’C1 and 30 u of topoisomerase I were performed for 30 min at 30°C in the presence and absence of Ca2+ and terminated with SDS. Lane 1, 5 mM-EDTA +0 PM-camptothecin; lane 2, 3 mM- CaCl, + 0 PM-camptothecin; lane 3, 5 mM-EDTA f 10 /AM-

camptothecin; lane 4, 3 mM-CaCl, + 10 PM-camptothecin. The arrow indicates cleavage at the recognition sequence.

Sequence-dependent Effect of Camptothecin in Vitro 339

Incubation of enzyme and DNA in the absence of (:a’+ and camptothecin did not cause cleavage upon addition of SDS (lane I), while incubation in Ca2+ resulted in cleavage at the recognition sequence (lane 2). When camptothecin was added to the incubation mixture cleavage was observed at some of the class B sites even in the absence of Ca2 + (e.g. position b, lane 3). In the presence of Ca2 ’ there was marked cleavage at the recognition sequence in addition to the cleavages at class B sites (lane 4). These observations demonstrate a

(a)

general stimulation of cleavage by Ca2’, although the degree of stimulation may vary at the different sites.

(e) The dissociation kinetics of cleavable complexes are affected differently by camptothecin

To investigate the stability of preformed cleavable complexes in the presence and absence of camptothecin, dissociation kinetic studies were done in 0.6 M-NaCi at 20°C. The dissociation

1 ,

5 IO 15 20 25 30 Time (min)

(b)

Figure 6. Dissociation kinetics of cleavable complexes. Two parallel 240-~1 reactions, each containing 24 fmol of HindIII-PvuII restriction fragment of pNC1 and 240 u of topoisomerase I, were preincubated at 30°C with or without 10 PM-captothecin. After 29.5 min a 30-~1 sample of each series was taken to 1 y0 SDS (final concn). At 30 min the remainder of the untreated reaction mixtures were quickly chilled to 20°C and adjusted to 0.6 M-NaCl (final concn). Samples (30 ~1) were taken at different times after NaCl addition and rapidly mixed with 6 ~1 6% SDS and processed for sequencing gels. (a) Lanes 1 to 8, no camptothecin; lanes 9 to 16, 10 FM-camptothecin. Lanes 1 and 9, 29.5 min preincubation and 1 yo SDS; lanes 2 to 8 and 10 to 16, time elapsed after adjusting to 0.6 M-NaCl; lanes 2 and 10: 0.5 min; lanes 3 and 11, 1 min; lanes 4 and 12, 5 min; lanes 5 and 13, 10 min; lanes 6 and 14, 20 min; lanes 7 and 15, 30 min; lanes 8 and 16, 60 min. The arrow indicates the position of cleavage at the hexadecameric sequence. (b) In each of the experimental series, the cleavage frequency at the recognition sequence or at position b (cf. Fig. 4) in any point were expressed as a percentage of the cleavage frequency in the sample taken after 29.5 min of preincubation. This percentage, the residual cleavage, was plotted against the time of sampling. The quantifications are based on densitometric scanning of the autoradiogram shown in (a). (0) Residual cleavage frequency at the recognition sequence in absence of camptothecin; (0) residual cleavage frequency at the recognition sequence in presence of camptothecin; (A) residual cleavage frequency at position b in the presence of camptothecin.

E. Kjeldsen et al.

kinetics were followed by treating samples removed at different times with SDS. The electrophoretic analysis of the resulting DNA fragments is shown in Figure 6. The enzyme-DNA complex formed at the recognition sequence dissociates rapidly in the presence of salt (Fig. 6(a), lanes 1 to 8) and is at the given temperature only marginally affected by camptothecin (Fig. 6(a), lanes 9 to 16). The dissociation kinetic of the enzyme-DNA complex formed at site b (cf. Fig. 4) is very different from that at site a (Fig. 6(a)). To quantify the differences, the cleavage frequencies were determined as described in Materials and Methods. 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 addition of salt, was plotted as a function of the time elapsed after addition of NaCl (Fig. 6(b)). The residual cleavage at the recognition site was reduced to 1 y0 after O-5 minute and cleavage was not detectable at later times. The complex formed at this site in the presence of camptothecin decayed at a slightly reduced rate. In contrast, the complex formed at position b in the presence of camptothecin decayed very slowly indeed. Thus, camptothecin exerts a

different stimulation of cleavage depending on the underlying DNA sequence and this seems to be due to a selective change in the kinetic stability of the enzyme-DNA complexes in question.

The increased cleavage frequency at position b observed after O-5 minute is probably due to a decreased masking effect of cleavage at position a, supporting the idea that the residual cleavage at position a decreases much more rapidly than that at position b. In addition, the observation demonstrates that multiple cleavages on the same fragment can occur within I6 bases.

(f) Characterization of potential human topoisomerase I DNA cleavage sites

Comparison of the underlying DNA sequences at the three classes of sites might provide some explanation for the differential effect of camptothecin on cleavage. To determine the DNA sequences of the various cleavage sites, DNA cleaved by topoisomerase I in the presence and absence of camptothecin was analyzed on sequencing gels in parallel with Maxam-Gilbert chemical degradation reactions of the same restriction fragment. Table 1 summarizes the

Table 1 Characterization of potential human topoisomerase I cleavage sites

Relative degree of cleavage

Position Sequence

5’-+3 Without With

B R CPT CPT

AGACTT AGAAAAATTT (a)t T A ++++ +++t TGTAGT AGGTTGAGGC (d) T A + +

TC(!CTT ATGCGACTCC (f) T A + GCCGTT GAGCACCGCC (c) 1 G AATGGT GCATGAAAAA (h) T G (+‘, CAGGGT GACGGTGCCG T G + GGTGAC GGTGCCGAGC TGACGG TGCCGAGGA;

C G + 0 T t

GACGGT GCCGAGGATG , T c + GAGGAT GACGATGAGC T G + GATGAC GATGAGC GC A (’ (: + GACGAT GAGCGCATTG T x C + CGCATT GTTAGATTTC T (: +

++ +++ ++t+ ++++ +t ++ ++ ++++ ++ ++ i-t

143 CTGCAT TAGGAAGCAG (e) T 1 + -

149 ATAACC AAGCCTATGC C A + -

92 GTGACG GTGCCGAGGA G G + -

67 GCGCAT TGTTAGATTT T T + -

The isolated 1.6 kb HindIII-PvuII 3’-end-labeled fragments were subjected to Maxam-Gilbert chemical degradation. For cleavage, 3 fmol of 3’-end-labeled fragments was incubated with SO units of human topoisomerase I in the absence or presence of camptothecin (10 PM, final concn). The reactions were terminated with SDS and the samples processed as described in Materials and Methods for co- electrophoresis with the Maxam-Gilbert reactions. The obtained sequences are listed. The position of the nucleotide that covalently binds the enzyme is indicated counting from the 3’ end label of the fragment. The relative degree of cleavage was determined by comparison of the frequency of cleavage at the indicated positions. +, relative degree of cleavage; -, no cleavage; B, the 3’ nucleotide that covalently binds the enzyme; R, the 5’ nucleotide that participates in religation; CPT, camptothecin.

t The letters in parentheses indicate the positions of cleavage as depicted in Fig. 4.

Sequence-dependent Effect of Camptothecin in Vitro 341

sequencing data from the experiments and correlates these data with the relative degree of cleavage obtained in the presence and absence of camptothecin. A number of potential cleavage sites that were not resolved in the gels shown in the previous Figures are also analyzed and included in the Table. It is not possible to deduce a consensus for the three classes of cleavage sites, although common features do exist. The 3’ nucleotide that becomes covalently attached to the eukaryotic topoisomerase I during enzyme action is a pyrimidine with a clear preference for a T residue in 99% of the cases. The 5’ nucleotide that participates in the religation reaction of the enzyme is a purine with a clear preference for a G residue in 90% of the cases.

4. Discussion

Eukaryotic topoisomerase I catalysis is inhibited by the antitumor drug camptothecin (Hsiang et al., 1985; Andoh et al., 1987). We have examined the effect of the drug on human topisomerase I DNA cleavage at the sequence level. Our results show that the enhancement of the overall topoisomerase I DNA cleavage elicited by camptothecin is accompanied by an alteration in the relative distribution of enzyme-substrate complexes on DNA. Thus, three classes of cleavable complexes can be distinguished using the drug in vitro. (1) Topoisomerase cleavage at class A sites, which include the hexadecameric recognition sequence, is only slightly stimulated by camptothecin. The stimulated cleavage is correlated with the slightly enhanced salt stability of the complexes and is not a result of an altered affinity of the enzyme for DNA, as the equilibrium dissociation constant remains unaltered. (2) Topoisomerase cleavage at the class B sites is stimulated up to 200-fold by camptothecin and the stability of the enzyme-DNA complexes to salt is greatly enhanced, indicating that the K, might be decreased in the presence of camptothecin. (3) Topoisomerase cleavage at class C sites is observed only at a high molar ratio of enzyme to DNA. The cleavage at these sites disappears when camptothecin is added, indicating that the drug might weaken the interaction between enzyme and DNA at these sequences.

The influence of CaZi and camptothecin on topoisomerase-I-mediated DNA cleavage was examined. Ca2+ is known to stimulate topoisomerase-I-catalyzed relaxation of supercoiled DNA, whereas camptothecin inhibits this reaction. Thus, the molecular mechanisms responsible for the observed accumulation of cleavable complexes in the presence of either Ca” or campt#othecin differ. Our data indicate that Ca2+ favors the formation of cleavable complexes and that camptothecin inhibits the dissociation of these complexes. The camptothecin-induced altered pattern in the relative distribution of topoisomerase I DNA cleavage sites and the differently enhanced kinetic stability cannot solely be explained by a simple

inhibition in the religation half-reaction, as proposed by Hsiang et al. (1985). Thus, the proposed model must be extended to include the fact that the effect is a sequence-dependent inhibition in the religation half-reaction. However, the observation that DNA cleavage at certain sites (class C) disappears in the presence of camptothecin indicates that additional mechanisms might be involved.

Comparison of the DNA sequences at the various cleavage sites does not reveal any obvious sequence homology, except for the similarities between the nucleotides directly adjacent to the cleavage site. At class B sites the 3’ nucleotide that transiently becomes covalently bound to the enzyme is found to be a pyrimidine with a clear preference for a T residue in 90% of the cases. At class A sites a similar T residue is present, as in the hexadecameric recognition motif. The preference for this base might be correlated with the high specificity in formation of the transient phosphodiester bond between the 3’ phosphoryl group and a tyrosyl residue of the enzyme (Champoux, 1981). At class B sites the 5’ nucleotide that participates in the religation half-reaction is found to be a purine with a clear preference for a G residue in 99% of the cases, while in the class A sites the preference is found to be an A residue. The sequencing data obtained from the cleavage sites that are eliminated in the presence of camptothecin (class C) do not give rise to any significant homology. Taken together the sequences at the three different classes of cleavage sites (Table 1) agree with the degenerate consensus derived by Edwards et al. (1902), except that G residues seem to be over-representled at the position of the 5’ nucleotide at the cleaved sites strongly stimulated by camptothecin (class B).

The sensitivity to camptothecin of topoisomerases isolated from evolutionarily distant species differs. In contrast to previous studies from the lower eukaryote Tetrahymenu (Thomsen et al., 19871, we find that the human enzyme responds strongly (up to 299fold) to the presence of camptothecin. Furthermore, it has been shown that camptothecin is unable to inhibit the relaxation activity of topoisomerase I isolated from yeast (T. Andoh, personal communication).

Previously we have shown that the hexadecameric recognition sequence functions as a good substrate for DNA cleavage and catalytic DNA breakage-reunion by eukaryotic topoisomerase I (Busk et al., 1987). Our present data show that camptothecin causes additional sites to be cleaved as efficiently as the hexadecameric recognition sequence, while the drug only affects this sequence slightly. These observations have the important implication that the cleavage intensities found in the presence of camptothecin do not necessarily reflect the rate of topoisomerase I catalysis at the various sites in the absence of the drug.

In conclusion, the data demonstrate that camptothecin has a dramatic sequence-dependent i’,

E. Kjeldsen et al.

effect) on human topoisomerase I DNA cleavage in vitro. Provided that camptothecin affects topoisomerase I-DNA interaction in viwo in the same manner as observed in vitro, our results represent a note of caution for the use of camptothecin in mapping of topoisomerase I binding sites, in vivo or in situ.

We thank Dr Steffen Junker for culturing the Daudi cells, Dr H.-P. Vosberg for the kind gift of polyclonal rabbit IgG directed against calf thymus topoisomerase I. Dr Rolf Lillebssk for doing the Western blot analysis, Drs Anni H. Andersen, Kent Christiansen and Ole F. Nielsen for helpful discussions, MS K. Andersen and B. Dal1 for skilful technical assistance, and MS Andrea K. Madsen for typing the manuscript. This study was supported by the Commission of the European Communities (contract B-6-0170-DK), the Danish Medical Research Council (grant no. 5.10.17.06) the Danish Cancer Society (grant nos 86-178, 87-025, and 87-088) the National Agency of Technology (contract no. 1985-133/001-85.521), and the Danish Programme of Biotechnology.

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Edited by P. Chambon