Báo cáo Y học: SF2/ASF protein inhibits camptothecin-induced DNA cleavage by human topoisomerase I potx

SF2/ASF protein inhibits camptothecin-induced DNA cleavage by human topoisomerase I Barbara Kowalska-Loth, Agnieszka Girstun, Agnieszka Piekiełko and Krzysztof Staron ´ Institute of Biochemistry, Warsaw University, Warsaw, Poland A splicing factor SF2/ASF is a natural substrate for the kinase activity of human topoisomerase I. This study dem- onstrates that SF2/ASF inhibits DNA cleavage by human topoisomerase I induced by the anti-cancer agent campto- thecin. The inhibition is independent of the phosphorylation status of SF2/ASF. We show that the inhibition did not result from binding of SF2/ASF to DNA that would hinder interactions between topoisomerase I and DNA. Neither it was a consequence of a loss of sensitivity of the enzyme to camptothecin. We provide evidence pointing to reduced formation of the cleavable complex in the presence of SF2/ ASF as a primary reason for the inhibition. This effect of SF2/ASF is reflected by inhibition of DNA relaxation catalysed by topoisomerase I. Keywords: topoisomerase I; SF2/ASF; camptothecin. Eukaryotic topoisomerase I (topo I) catalyses DNA relax- ation and plays a key role in DNA replication, transcription and recombination in the cell [1]. Topo I is also a target for several anti-cancer drugs derived from the cytotoxic plant alkaloid camptothecin [2]. A transient intermediate of the enzyme’s catalytic cycle is the cleavable complex, consisting of the enzyme linked covalently to one strand of DNA. This complex is stabilized by camptothecin and can be detected as topo I-associated DNA single-strand breaks [3]. These breaks are usually called camptothecin-induced DNA cleavage. Camptothecin-induced DNA cleavage is essen- tially reduced by binding of SV40 T antigen [4] or ATP [5] to topo I. The complex is not detected for several mutated forms of the enzyme that are resistant to camptothecin (reviewed in [6]). Sensitivity to camptothecin is also dimini- shed by dephosphorylation of topo I [7,8]. Human topoisomerase I (htopo I) possesses a protein kinase activity which is specific towards serine residues of splicing factors containing a serine-arginine (SR) motif [9]. Phosphorylation of SR proteins is instrumental for the recruitment of these proteins to active sites of transcription in vivo [10] and for their activity as splicing factors [11]. SF2/ ASF is the main splicing factor containing an SR motif [12] phosphorylated by htopo I/kinase [9]. The binding site for SF2/ASF in htopo I is located between residues 135 and 175 [13]. This region is included in the N-terminal domain of the enzyme that in general is dispensable for both relaxation and binding of camptothecin [14,15]. However, detailed studies have recently revealed that the N-terminal domain influences the rate of relaxation and sensitivity of the enzyme to camptothecin [16]. In the present work we report that the natural substrate for the kinase activity of htopo I, SF2/ASF protein, inhibits camptothecin-induced DNA cleavage by the enzyme. This effect results from reduced amount of the cleavable complex formed by htopo I in the presence of SF2/ASF. MATERIALS AND METHODS Plasmids and strains The pRS426-based plasmid, containing complete human topo I cDNA under the control of the GAL1-10, was a gift from B. R. Knudsen (University of Aarhus, Denmark). The yeast strain EKY3 (Dtop1)wasagiftfromJ.Fertala (Thomas Jefferson University, Philadelphia, USA) and was used to express htopo I. The plasmid containing ASF-1 cDNA was a gift from J. Tazi (Universite ´ Montpellier II, France). SF2/ASF was expressed from this plasmid in Escherichia coli strain TG1. pQE32 was provided by Qiagen. pBR322 was from the laboratory collection. pBR322 and pQE32 were produced in E. coli strain DH5a. Expression and purification of proteins The yeast cells were transformed with the expression plasmid containing htopo I cDNA. Expression of htopo I was induced in 2-L cultures with 2% galactose. After 4 h cells were harvested, washed once with 1 M sorbitol, 25 m M EDTA, pH 8.0, 50 m M dithiothreitol and lysed by addition of zymolyase 100T (5 mgÆmL )1 ; from Seikagaku) in 1 M sorbitol. Centrifuged spheroplasts were frozen and then extracted twice with 20 m M Tris, pH 7.5, 0.5 M KCl, 1 m M EDTA, 1 m M dithiothreitol, 10% glycerol, 1 m M phenyl- methanesulfonyl fluoride. The extracts were then purified by successive heparin-agarose and nickel-nitrilotriacetic acid- agarose chromatography, as described previously [17]. Coomassie staining of the purified preparation indicated an  91-kDa polypeptide as the only band on SDS polyacrylamide gels. The band was recognized by Correspondence to K. Staron ´ , Institute of Biochemistry, Warsaw University, ul. Miecznikowa 1, 02–096 Warszawa, Poland. Fax: + 48 225543116, Tel.: + 48 225543114, E-mail: staron@biol.uw.edu.pl Abbreviations: topo I, topoisomerase I; htopo I, human topoisomerase I. Enzymes: DNA topoisomerase I (EC 5.99.1.2). (Received 18 February 2002, revised 20 May 2002, accepted 31 May 2002) Eur. J. Biochem. 269, 3504–3510 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03037.x scleroderma antibody to human topo I (TopoGEN). Relaxing activity of the enzyme was 6.1 · 10 6 ±2.2· 10 6 (n ¼ 4) units per mg protein. Htopo I was also active as a protein kinase and selectively phosphorylated the SF2/ASF substrate. The htopo I preparations were adjusted to 20% glycerol and stored at )80 °C. Bacteria transformed with the expression vector for SF2/ASF were grown in 100 mL cultures. Expression of the protein was induced by 1 m M isopropyl thio-b- D -galactoside for 1 h. SF2/ASF was isolated and purified as described previously [9] and stored at )80 °C. Directly before each experiment, SF2/ASF was centrifuged to remove aggregates. The phosphorylated form of SF2/ASF was produced by the in vitro phosphorylation of isolated SF2/ASF and then was directly used in the cleavage assay. To phosphorylate SF2/ASF, htopo I and unlabeled 1 m M ATP were used under the conditions of the kinase assay [9]. When the mixture was further used in the cleavage assay, it was diluted so that concentration of ATP was 150 l M . At this concentration, ATP did not influence DNA cleavage (not shown). Based on analysis of the htopo I-catalysed incorporation of the label from [c- 32 P]ATP to samples of SF2/ASF previously either phosphorylated or subjected to the mock procedure, we calculated that 87% of the accessible sites were phos- phorylated in the preparation used in the cleavage assay as Ôphosphorylated SF2/ASFÕ. The amount of htopo I and SF2/ASF was quantified by densitometric scanning of SDS-polyacrylamide gels using BSA as a standard. DNA cleavage assay Routinely, the DNA substrate labelled at its 3¢ end was used in the cleavage assay. The 505-bp DNA fragment was prepared from pQE32 vector (Qiagen). The vector DNA was cleaved with EcoRI and labeled using [a- 32 P] ATP (3000 CiÆmmol )1 ; Amersham) and T7 sequenase (Amer- sham) according to the manufacturer’s protocol. The DNA sample was then cleaved with DraIandthefragmentof 505 bp was purified by an agarose gel electrophoresis. When indicated, DNA labelled at 5¢ end was used. The 653-bp fragment was prepared from pBR322 plasmid cleaved with EcoRI and SalI. The EcoRI–SalI fragment was purified by agarose electrophoresis, dephosphorylated by calf intestinal alkaline phosphatase (USB) and labeled using T4 polynu- cleotide kinase and [c- 32 P]ATP (5000 CiÆmmol )1 ,Amer- sham) [18]. The cleavage reaction was performed with 0.15–2.5 ng of labeled DNA substrate and an appropriate amount of htopo I in the buffer containing 50 m M KCl, 10 m M MgCl 2 ,0.1m M EDTA, 50 lgÆmL )1 gelatin, 20 m M Tris, pH 7.5 and various amounts of SF2/ASF. When indicated, 1 lL of camptothecin dissolved in dimethylsulfoxide was in the final reaction volume 100 lL. The mixtures were incubated for 30 min at 30 °C. The reaction was stopped by addition of 12 lL of 10% SDS and samples were incubated for 15 min at 75 °C, chilled and digested for 1 h at 37 °C with proteinase K. When indicated, the protein digestion step was omitted but instead 1 m M phenyl- methanesulfonyl fluoride was added. A carrier DNA was added before ethanol precipitation. The DNA samples were dissolved in 10 m M Tris containing 25% formamide, heated to 75 °C for 4 min and analysed using 7.5% polyacrylamide/7 M urea gels. Gels were exposed for 24 h using Rentgen XS-1 (Foton) or Biomax MS (Kodak) films. Mobility shift assay The mobility shift assay was performed using a 377-bp EcoRI–BamHI fragment of pBR322, labelled at the 3¢ end as described above. Purified proteins were incubated with 1.69 fmol DNA in 40 m M Tris/acetate, 1 m M EDTA, pH 8.0. The DNA and DNA–protein complexes were run on 6% native polyacrylamide gel for 1 h at 200 V. Gels were exposed for 24 h using Rentgen XS-1 (Foton). Relaxation assay DNA relaxation activity of htopo I was measured using supercoiled pBR322 plasmid DNA as a substrate. Reaction mixtures (10 lL final volume) contained 0.2–0.4 lg plasmid DNA, 120 m M KCl, 10 m M MgCl 2 ,0.1m M EDTA, 10 m M 2-mercaptoethanol, 100 lgÆmL )1 BSA, 40 m M Tris/HCl, pH 7.5 and various amounts of htopo I protein. When indicated, the mixtures contained various amounts of SF2/ ASF. Reaction was performed either at 30 °C(forestima- tion of specific activity of htopo I) or at 0 °Candwas terminated by addition of 5 lL of the stop buffer (27% sucrose, 0.67% SDS, 67 m M EDTA, pH 8, Orange G). The samples were loaded onto a 1% agarose gel and subjected to electrophoresis for 1.5 h at 100 V. Gels were stained with ethidium bromide and the reaction was quantified by densitometric scanning of negatives of photographed elec- trophoretic gels. Relaxation activity was calculated from decrease of the amount of supercoiled DNA. One unit relaxed 50% of the substrate DNA after 30 min at 30 °C. Kinase assay Htopo I kinase activity was measured using SF2/ASF and [c- 32 P]ATP (5000 CiÆmmol )1 ; Amersham) as substrates, under conditions described previously [9]. The proteins were analysed using 12% SDS/polyacrylamide gels, dried and exposed for 24 h using Rentgen XS-1 (Foton) films. RESULTS Inhibition of the DNA cleavage by SF2/ASF To study camptothecin-induced DNA cleavage by htopo I we routinely used the EcoRI–DraI fragment of pQE32 DNA labelled at the 3¢ end. The cleavage was visible at the concentration of camptothecin as low as 1 l M . However, to study effects of SF2/ASF on the cleavage we used camp- tothecin concentration of 100 l M (Fig. 1). This was to avoid a possibility of lowered drug-sensitivity for the enzyme remaining in the complex with SF2/ASF. Such an effect, appearing as an elevated concentration of camptothecin needed to induce DNA cleavage, has been previously observed for some point mutations in htopo I [19]. We examined dependence of the inhibition of DNA cleavage on the amount of SF2/ASF present in the reaction mixture. We performed the cleavage assay with a fixed amount of htopo I and various amounts of SF2/ASF. An example of such an experiment is shown in Fig. 2. Ó FEBS 2002 SF2/ASF and effect of camptothecin (Eur. J. Biochem. 269) 3505 Approximately 50% inhibition of the cleavage was achieved at the htopo I: SF2/ASF ratio of 1.79 ± 0.76 (± SD, n ¼ 8). The recombinant SF2/ASF protein used in the experi- ments described above remained unphosphorylated within its SR motif recognized by htopo I [13]. To investigate the effect of phosphorylation of SF2/ASF, we used SF2/ASF previously phosphorylated by htopo I. We found that phosphorylated SF2/ASF inhibited DNA cleavage 1 to the same extent as the unphosphorylated protein (not shown). Looking for a possible reason for inhibition of campto- thecin-induced DNA cleavage by SF2/ASF we considered three possibilities. (a) The effect of SF2/ASF results not from its interaction with htopo I but from its binding to DNA. (b) SF2/ASF decreases the sensitivity of htopo I to camptothecin. (c) SF2/ASF inhibits the formation of the cleavable complex by htopo I in the absence of camptothe- cin. These possibilities were successively examined in the further work. Accessibility of DNA for htopo I Unphosphorylated, recombinant SF2/ASF is a basic pro- tein and is known to bind nucleic acids nonspecifically [20]. Thus, a trivial explanation for the inhibition of the DNA cleavage could be that SF2/ASF preferentially bound to DNA and prevented htopo I from contacting with the substrate. To check this possibility we performed two sets of experiments. The first one was to indicate whether htopo I or SF2/ASF binds to DNA more effectively. The other was to check whether SF2/ASF blocked accessibility of htopo I to DNA. To follow binding of proteins to DNA we employed a mobility shift assay (Fig. 3). Under these conditions, a portion of DNA was shifted towards slower migrating band if 0.14 pmol of htopo I was present in the sample (Fig. 3A). At higher amounts of htopo I (0.28 pmol), all DNA present in the sample aggregated and did not penetrate into the gel (Fig. 3B, lane 2). The mobility shift did not indicate DNA– protein complexes for SF2/ASF protein. Instead, we observed aggregation of DNA (Fig. 3B, lane 5). An amount of SF2/ASF at which the aggregation occurred (1.58 pmol) was more than five times higher than that needed for the same effect caused by htopo I. Because an effective inhibition of camptothecin-induced DNA cleavage was observed at an  twofold excess of SF2/ASF over htopo I (Fig. 2), the mobility shift experiments excluded a possibility that the inhibition resulted from removal of DNA aggre- gated by SF2/ASF. However, these experiments alone did not give answer to the question whether SF2/ASF can block accessibility of htopo I to DNA. To check accessibility of DNA to proteins in the presence of htopo I, SF2/ASF and both proteins we employed restriction nucleases as reference probes. To exclude any specific interaction between the endonuclease and examined proteins we used three different restriction endonucleases: HindIII, RsaIandSphI, observing the same pattern of digestion in each case. Results obtained with HindIII and RsaI are presented in Fig. 4. Each of the restriction endonucleases employed in the assay cut the substrate (the EcoRI–DraI fragment of pQE32 DNA) only once. Diges- tion with endonucleases was performed under conditions of the cleavage assay in the presence of camptothecin. Such an Fig. 2. Dependence of the inhibition on SF2/ASF concentration. 3¢- Labelled DNA fragment was a substrate. The concentration of camptothecin was 100 l M ; 0.65 pmol htopo I and various amounts of SF2/ASF were used. Lane 1, DNA alone; lane 2, htopo I; lanes 3–8, htopo I + SF2/ASF. Amounts of SF2/ASF were: lane 3, 0.2 pmol; lane 4, 0.4 pmol; lane 5, 0.8 pmol; lane 6, 2 pmol; lane 7, 4 pmol; lane 8, 8 pmol. Fig. 1. Inhibition of camptothecin-induced cleavage by SF2/ASF. 3¢-Labelled DNA fragment was a substrate. The concentration of camptothecin was 100 l M ;0.35pmolhtopoIand0.8pmolSF2/ASF wereused.Lanes:1,DNAalone;2,DNA+camptothecin; 3,DNA+htopoI;4,DNA+htopoI+camptothecin;5,DNA+ htopo I+ SF2/ASF; 6, DNA +htopo I +camptothecin +SF2/ASF. 3506 B. Kowalska-Loth et al. (Eur. J. Biochem. 269) Ó FEBS 2002 approach had an advantage of assuring that the amount of htopo I used in the test was big enough to cleave DNA and the concentration of SF2/ASF was high enough to inhibit htopo I-catalysed cleavage. In this case, the rate of compe- tition between endonucleases and htopo I was not clear because DNA fragments produced by the endonucleases were further cleaved by htopo I (Fig. 4, lanes 2 and 6). However, it is clearly visible that SF2/ASF only slightly inhibited the cutting of DNA by either HindIII or RsaI. At thesameconcentrationofSF2/ASFinthepresenceof htopo I, both endonucleases cut DNA less efficiently than in the presence of SF2/ASF alone (compare lanes 3 and 4 or 7 and 8) indicating that SF2/ASF did not block accessibility of htopo I to DNA. Sensitivity of the kinase reaction of htopo I to camptothecin Another explanation for the inhibition of DNA cleavage by SF2/ASF would be lack of binding of camptothecin to existing cleavable complex. This, however, was not a case because htopo I remained sensitive to camptothecin in the presence of SF2/ASF. We examined it for the kinase reaction that used SF2/ASF as a substrate. Under condi- tions of this reaction SF2/ASF was present in the eightfold excess over htopo I. Addition of increasing amounts of DNA to the reaction mixture resulted in a gradual decrease and eventually a complete inhibition of phosphorylation of SF2/ASF by htopo I (Fig. 5A). If small amount of DNA was present in the reaction mixture (0.15 fmol pBR322 DNA), the inhibi- tion of the kinase reaction by DNA was significantly enhanced upon subsequent addition of camptothecin (Fig. 5B). DNA relaxation by htopo I performed in the presence of SF2/ASF remained sensitive to camptothecin (not shown). Inhibition of formation of the cleavable complex by SF2/ASF in the absence of camptothecin A likely explanation for the inhibition of DNA cleavage is that SF2/ASF directly influenced the reaction catalysed by htopo I so that a reduced amount of the covalent DNA– htopo I complex was formed. To check this possibility we performed the cleavage reaction without camptothecin. Because of the low efficiency of the cleavage occurring in the absence of camptothecin we used the substrate labelled at its 5¢ end that allowed us to distinguish accidental DNA breaks from those introduced by htopo I. The product of the cleavage of the 5¢-labelled substrate by htopo I would be the enzyme covalently bound to the labelled strand. Such a cleavage product would enter gel only after prior digestion Fig. 3. Binding of htopo I and SF2/ASF to DNA revealed by mobility shift assay. (A) Binding of htopo I to DNA. Lanes: 1, DNA alone; 2, DNA + 0.14 pmol htopo I. (B) Aggregation of DNA by htopo I and SF2/ASF. Lanes: 1, DNA alone; 2, DNA + 0.28 pmol htopo I; 3, DNA + 0.28 pmol SF2/ASF, 4, DNA + 0.56 pmol SF2/ASF, 5, DNA + 1.58 pmol SF2/ASF; 6, DNA + 0.28 pmol htopo I +0.56 pmol SF2/ASF. Fig. 4. Accessibility of DNA for restriction endonucleases in the pres- ence of htopo I, SF2/ASF or both proteins. 3¢-Labelled DNA fragment was a substrate. The concentration of camptothecin was 100 l M ; 0.92 pmol htopo I, 0.92 pmol SF2/ASF, 10 U HindIII and 15 U RsaI were used. The mixtures were incubated for 30 min at 30 °C. Lanes: 1, HindIII; 2, HindIII + htopo I; 3, HindIII + SF2/ASF; 4, HindIII + htopo I + SF2/ASF; 5, RsaI; 6, RsaI + htopo I; 7, RsaI+SF2/ASF; 8, RsaI + htopo I + SF2/ASF. Ó FEBS 2002 SF2/ASF and effect of camptothecin (Eur. J. Biochem. 269) 3507 with proteinase that removed the bound protein [21]. Figure 6 shows the cleavage pattern for htopo I acting on 5¢-labelled DNA in the absence of camptothecin. Bands absent in the undigested sample but appearing upon digestion with proteinase K were identified as resulting from htopo I cleavage. SF2/ASF abolished DNA cuts introduced by htopo I in the absence of camptothecin. The effect of SF2/ASF was specifically restricted to bands coming from htopo I activity. This observation pointed to smaller amount of covalent DNA–htopo I complex formed in the presence of SF2/ASF as a primary reason for the inhibition of camptothecin-induced DNA cleavage. Effect of SF2/ASF on DNA relaxation The question we wished to answer next was whether the reduced amount of the cleavage complex formed in the presence of SF2/ASF exerted any effect on DNA relaxation by htopo I. At a high substrate DNA/topo I ratio, usually used in relaxation tests, the release of the enzyme from DNA is a rate-limiting step for relaxation [22]. On the other hand, an impaired DNA cleavage observed in the cleavage assay should be reflected by inhibition of the reaction catalysed by htopo I rather than by inhibition of the movement of the enzyme between DNA substrate molecules. Elimination of a dissociation of htopo I as a rate-limiting step for relaxation can be achieved by decreasing the DNA/htopo I ratio during the relaxation to 1 : 1 or less [16]. However, in this case the reaction rate should be slowed because of an elevated amount of the enzyme [16]. We achieved this by reducing the reaction temperature to 0 °C. The reaction rate of relaxation was approximately 10–20 times lower at 0 °C than at 30 °C. Low temperature did not impair binding of SF2/ASF to htopo I because phosphorylation of this protein by htopo I as well as inhibition of camptothecin- induced DNA cleavage by SF2/ASF were still observed at 0 °C (not shown). Under conditions of the relaxation assay SF2/ASF slowed the relaxation of DNA (Fig. 7). Fig. 6. Inhibition of DNA cleavage by SF2/ASF in the absence of camptothecin. 5¢-Labelled DNA fragment was a substrate. 0.32 pmol htopo I and 1.3 pmol SF2/ASF were used. Lanes: 1, DNA alone; 2, htopo I, no proteinase K; 3, htopo I, digested with proteinase K; 4, htopo I + SF2/ASF, no proteinase K; 5, htopo I + SF2/ASF, digested with proteinase K. Fig. 5. Inhibition of kinase activity of htopo I by DNA and campto- thecin. htopo I (0.16 pmol) and SF2/ASF (1.25 pmol) were used. The concentration of camptothecin was 100 l M ; pBR322 DNA was used to inhibit the reaction. The reaction was carried out for 8 min at 30 °C. (A) Inhibition by DNA. Lanes: 1, no DNA; 2, 0.15 fmol DNA; 3, 1.5 fmol DNA; 4, 15 fmol DNA. (B) Effect of camptothecin at 0.15 fmol DNA. Lanes: 1, camptothecin, 2, DNA; 3, DNA + camptothecin. Fig. 7. Effect of SF2/ASF on relaxing activity at the equimolar ratio of htopo I and DNA. 70 fmol pBR322 DNA, 71 fmol htopo I and 71 fmol SF2/ASF were used. The reaction was carried out at 0 °C. Lanes: 1, DNA alone; upper gel, DNA + htopo I; lower gel, DNA + htopo I + SF2/ASF. The reaction times were: lane 2, 15 s; lane 3, 30 s; lane 4, 1 min, lane 5, 2.5 min; lane 6, 5 min; lane 7, 10 min; lane 8, 20 min. 3508 B. Kowalska-Loth et al. (Eur. J. Biochem. 269) Ó FEBS 2002 DISCUSSION The results presented in this work show that the substrate for the kinase activity of htopo I, SF2/ASF, inhibits the camptothecin-induced DNA cleavage. Looking for a primary reason for this phenomenon we considered: (a) a direct effect of SF2/ASF on DNA, (b) loss of sensitivity to camptothecin for htopo I, or (c) a direct effect of SF2/ASF on the reaction catalysed by htopo I. We excluded first two possibilities and provided evidence pointing to the latter one. Modulation of relaxing activity by a direct effect of ssb proteins on DNA has been reported for bacterial topo I [23]. We took into consideration the possibility that SF2/ ASF acted directly on DNA because it had previously been reported to bind nonspecifically to nucleic acids [21]. SF2/ ASF could thus preferentially bind to DNA and prevent this way DNA from cutting by htopo I. We excluded this possibility showing that htopo I bound to DNA more effectively than SF2/ASF and that SF2/ASF did not block accessibility of htopo I to DNA. A decrease in sensitivity to camptothecin has previously been observed for several point mutations in htopo I [6]. However, it has also been shown that htopo I is sensitive to camptothecin in the presence of the excess of SF2/ASF because the drug inhibits the kinase reaction that uses SF2/ ASF as a substrate [9]. In this work we determined results similar to [9]. They 2 ruled out the possibility that a reason for camptothecin-induced DNA cleavage by SF2/ASF is a loss in sensitivity of htopo I to camptothecin. The question that still remains to be answered is why camptothecin inhibits the kinase reaction if SF2/ASF essentially reduces the amount of the cleavable complex formed in the presence of the drug. A helpful indication could be the observation that inhibition of the kinase reaction does not need camptothecin but is achieved by immobilization of htopo I on DNA, either by noncovalent binding or in the form of the cleavable complex. One might thus speculate that, if SF2/ASF inhibits the reaction catalysed by htopo I (see below), stabilization of a rarely appearing cleavable complex by camptothecin could shift the equilibrium between htopo I engaged in phosphorylation and that bound to DNA towards the latter pool. Finally, we directly showed that SF2/ASF specifically inhibits formation of the cleavable complex by htopo I in the absence of camptothecin. A reason for this phenomenon could be that either SF2/ASF prevents htopo I from binding to DNA or it impairs the reaction catalysed by the enzyme. The first mechanism has been revealed as underlying the inhibition of camptothecin-induced DNA cleavage by ATP [5]. On the other hand, a shorter half-time for the cleavable complex eventually resulting in its reduced amount, has been reported for reconstituted htopo I lacking the linker region [14]. Inhibition of the kinase reaction by DNA and competition experiments with endonucleases did not suggest that SF2/ASF diminishes binding of htopo I to DNA. Thus, a direct effect of SF2/ASF on the reaction catalysed by htopo I is a more likely mechanism. An inhibitory effect of SF2/ASF on camptothecin- induced DNA cleavage was observed for both the unmodi- fied and phosphorylated form of the protein. This observation is consistent with findings showing that the interaction of SF2/ASF with htopo I uses the RS domain of this protein and that phosphorylation of this domain does not impair the interaction [13]. A direct reason for cytotoxic effects of camptothecin is stabilization of the cleavable complex, eventually leading to DNA damage [24]. Because SF2/ASF is a natural substrate for htopo I kinase activity [9], inhibition of the formation of camptothecin-stabilized cleavable complex should be rele- vant to the in vivo conditions. An clear conclusion is that SF2/ASF might play a protective role against camptothe- cin-induced DNA damage in transcriptionally active regions of chromatin where the kinase activity of htopo I is exploited. A consequence of the reduced formation of the cleavable complex in the presence of SF2/ASF is the slowing of DNA relaxation. Such an effect was observed in this work for the reaction performed at the equimolar ratio of htopo I and the substrate DNA. It is consistent with effects of another substrate of the kinase reaction, ATP, which disable htopo I from binding to DNA [9]. It has been proposed that ATP switches the activity of htopo I from DNA relaxation on phosphorylation of SR proteins [9]. The results of this work show that SF2/ASF could work in concert with ATP as an inhibitor of DNA relaxation. ACKNOWLEDGEMENTS We wish to thank Dr Birgitte R. Knudsen from University of Aarhus, Denmark, Dr Jamal Tazi from Universite ´ Montpellier II, France and Dr Jolanta Fertala from Thomas Jefferson University, Philadelphia, USA, for providing plasmids and the yeast strain. We thank Alicja Czubaty for critical comments on the manuscript. This work was supported by the State Committee for Scientific Research (KBN, grant 6 P04A 069 15). REFERENCES 1. Wang, J.C. (1996) DNA topoisomerases. Annu. Rev. Biochem. 65, 635–692. 2. Rothenberg, M.L. (1997) Topoisomerase I inhibitors: review and update. Ann. Oncol. 8, 837–855. 3. Hsiang, Y.H., Hertzberg, R., Hecht, S. & Liu, L.F. (1985) Camptothecin induces protein-linked DNA breaks via mamma- lian topoisomerase I. J. Biol. Chem. 260, 14873–14878. 4. Pommier, Y., Kohlhagen, G., Wu, C. & Simmonds, D.T. (1998) Mammalian DNA topoisomerase I activity and poisoning by camptothecin are inhibited by simian virus 40 large T antigen. Biochemistry 37, 3818–3823. 5. Chen, H.J. & Hwang, J. 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