Functional interactions of DNA topoisomerases with a human replication origin
2007; Springer Nature; Volume: 26; Issue: 4 Linguagem: Inglês
10.1038/sj.emboj.7601578
ISSN1460-2075
AutoresGulnara Abdurashidova, Sorina Radulescu, Oscar Sandoval, Sotir Zahariev, M. Danailov, Alexander Demidovich, Laura Santamaria, Giuseppe Biamonti, Silvano Riva, Arturo Falaschi,
Tópico(s)Acute Lymphoblastic Leukemia research
ResumoArticle8 February 2007free access Functional interactions of DNA topoisomerases with a human replication origin Gulnara Abdurashidova Gulnara Abdurashidova Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Sorina Radulescu Sorina Radulescu Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Oscar Sandoval Oscar Sandoval Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Sotir Zahariev Sotir Zahariev Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Miltcho B Danailov Miltcho B Danailov Sincrotrone Trieste, Trieste, Italy Search for more papers by this author Alexander Demidovich Alexander Demidovich Sincrotrone Trieste, Trieste, Italy Search for more papers by this author Laura Santamaria Laura Santamaria Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Giuseppe Biamonti Giuseppe Biamonti Molecular Biology Section, Istituto di Genetica Molecolare, CNR, Pavia, Italy Search for more papers by this author Silvano Riva Silvano Riva Molecular Biology Section, Istituto di Genetica Molecolare, CNR, Pavia, Italy Search for more papers by this author Arturo Falaschi Corresponding Author Arturo Falaschi Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Molecular Biology Laboratory, Scuola Normale Superiore, Pisa, Italy Laboratory of Gene and Molecular Therapy, Istituto di Fisiologia Clinica, CNR, Pisa, Italy Search for more papers by this author Gulnara Abdurashidova Gulnara Abdurashidova Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Sorina Radulescu Sorina Radulescu Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Oscar Sandoval Oscar Sandoval Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Sotir Zahariev Sotir Zahariev Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Miltcho B Danailov Miltcho B Danailov Sincrotrone Trieste, Trieste, Italy Search for more papers by this author Alexander Demidovich Alexander Demidovich Sincrotrone Trieste, Trieste, Italy Search for more papers by this author Laura Santamaria Laura Santamaria Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Giuseppe Biamonti Giuseppe Biamonti Molecular Biology Section, Istituto di Genetica Molecolare, CNR, Pavia, Italy Search for more papers by this author Silvano Riva Silvano Riva Molecular Biology Section, Istituto di Genetica Molecolare, CNR, Pavia, Italy Search for more papers by this author Arturo Falaschi Corresponding Author Arturo Falaschi Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Molecular Biology Laboratory, Scuola Normale Superiore, Pisa, Italy Laboratory of Gene and Molecular Therapy, Istituto di Fisiologia Clinica, CNR, Pisa, Italy Search for more papers by this author Author Information Gulnara Abdurashidova1,‡, Sorina Radulescu1,‡, Oscar Sandoval1, Sotir Zahariev1, Miltcho B Danailov2, Alexander Demidovich2, Laura Santamaria1, Giuseppe Biamonti3, Silvano Riva3 and Arturo Falaschi 1,4,5 1Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 2Sincrotrone Trieste, Trieste, Italy 3Molecular Biology Section, Istituto di Genetica Molecolare, CNR, Pavia, Italy 4Molecular Biology Laboratory, Scuola Normale Superiore, Pisa, Italy 5Laboratory of Gene and Molecular Therapy, Istituto di Fisiologia Clinica, CNR, Pisa, Italy ‡These authors contributed equally to this work *Corresponding author. International Centre for Genetic Engineering and Biotechnology, ICGEB, Padriciano 99, Trieste I-34012, Italy. Tel.: +39 040 3757303; Fax: +39 040 3757353; E-mail: [email protected] The EMBO Journal (2007)26:998-1009https://doi.org/10.1038/sj.emboj.7601578 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation. Introduction The regulation of DNA replication of eukaryotic organisms is mediated by the cell-cycle-dependent assembly and reorganization of specific multiprotein complexes on the origins of DNA replication. DNA topoisomerases are required for the processes of activation of the ori C of Escherichia coli (Kornberg, 1984) and of the origins of SV40 (Halmer et al, 1998), BPV (Hu et al, 2006) and EBV genomes (Kawanishi, 1993); furthermore, in vitro DNA synthesis with a Saccharomyces cerevisiae system requires negatively supercoiled DNA and the action of DNA topoisomerase I (topo I) (Mitkova et al, 2005), and negatively supercoiled DNA is also required for the binding of the Drosophila ORC (Remus et al, 2004). Thus, the modifications of the specific protein–DNA interactions occurring at human replication origins may entail DNA topoisomerase-induced modulations of the topological state of the origin DNA. We addressed this question for the origin of DNA replication located at the 3′ end of the human gene for lamin B2, for which we have described the precise cell-cycle-modulated interactions with some components of the replicative complexes (Abdurashidova et al, 2003). The sites of action of topoisomerases may be detected in vivo by freezing specifically and reversibly the intermediates of the reaction they catalyze using specific poisons: camptothecin (CPT) for topo I and the etoposide VP16 for topo II; these drugs forbid the reformation of the cleaved phosphodiester bonds and leave the enzymes covalently bound to the 3′-phosphate (topo I) or 5′-phosphate (topo II) of the cleaved bond, freezing the so-called ‘cleavage complex’ (Burden and Osheroff, 1998; Pommier et al, 1998). By coupling the treatment with the appropriate poison with ligation-mediated PCR (LM-PCR) analysis (topo I; Strumberg et al, 2000; Mueller et al, 2001) or terminal transferase-dependent PCR (TD-PCR) analysis (topo II; Komura and Riggs, 1998), we localized the precise positions of the two topos in vivo, in a 1500-bp region comprising the lamin B2 origin, throughout the cell cycle. We also demonstrated that topo I and II are members of the human origin binding complex and established that topo I activity is essential for replicon firing. Furthermore, in a parallel in vitro approach, we observed different modes of interaction of topo I and II with the origin. Results and discussion Presence of active topo I and II at the origin To gain insights into the role of DNA topology for origin function, we investigated the behavior of topo I, which is required for origin function in viruses and yeast. To identify the possible presence of active topo I in the origin area, asynchronous HeLa cells were treated for 1 min with increasing concentrations of CPT. DNA was extracted and analyzed by LM-PCR with appropriate primers for the upper and lower strands (see Materials and methods). This analysis (Figure 1A) identifies the positions of the 5′OH residues arising from topo I action on either strand in the area covered in vivo by the replicative complexes. Only two sites are cleaved by the enzyme, one on the upper strand between nucleotides 3890 and 3891, and the other on the lower strand between nucleotides 3956 and 3957 (see Figure 1C). Figure 1.Interaction of topo I and II with the lamin B2 origin in vivo. (A) LM-PCR-mediated analysis of the topo I–DNA cleavage complexes induced by 1 min treatment of asynchronously growing HeLa cells with increasing concentrations of CPT (1, 10 and 100 nM, lanes 3–5 and 1 μM, lanes 6 and 12) or with 10 μM gimatecan (lane 7). Lanes 2 and 11: control genomic DNA from untreated cells; lanes 1 and 10: Maxam–Gilbert sequencing reaction. TD-PCR analysis of the distribution of UV photoproducts along the origin region in UV-irradiated HeLa cells treated for 1 min with 1 μM CPT (lanes 9 and 14) or left untreated (lanes 8 and 13). (B) TD-PCR-mediated analysis of the topo II–DNA cleavage complexes induced by treatment of asynchronously growing HeLa cells with 10 nM VP16 (lanes 3 and 10); lanes 2 and 9: control genomic DNA from untreated cells; DNA immunoprecipitated with anti-topo II antibody from cells subjected (lane 4) or not subjected (lane 5) to VP16 treatment; TD-PCR analysis of the distribution of UV photoproducts along the origin region in UV-irradiated HeLa cells treated for 1 min with 10 nM VP16 (lanes 7 and 12) or left untreated (lanes 6 and 11). (C) Summary of topo I and II in vivo cleavage sites at the lamin B2 origin area involved in the replicative complexes. Leading strand start sites are indicated by arrows and DNA cleavage complexes by filled triangles. Download figure Download PowerPoint To rule out the possibility that these cuts derive from secondary effects of the CPT treatment, such as a possible disruption of the chromatin and replicative complex structure, we performed a photo-footprinting analysis of the origin area in cells treated or not treated with CPT. Irradiation of the cells with short UV pulses using a femtosecond laser source induces DNA damage (photoproducts or protein–base crosslinks). As shown in lanes 8–9 and 13–14 of Figure 1A, the pattern of photo-footprinting on both strands was not perturbed by CPT, with the single conspicuous difference of a band related to the frozen topo I cleavage complex. The selection of the cleaved phosphodiester bond is an intrinsic property of the enzyme, as (as shown in lane 7 of Figure 1A) the same cleavage in vivo is detected upon treatment with gimatecan, a CPT derivative modified at the 7-carbon and displaying a different electronic structure. We also investigated the involvement of topo II. In the past, a topo II cleavage site was mapped in a 2-kb region containing the lamin B2 origin (Lagarkova et al, 1998). We treated asynchrounous HeLa cells with VP16 for 1 min and mapped the position of the topo II-mediated cleavage complexes by TDPCR. The results in Figure 1B show the interaction of topo II with two sites, both in the area covered by the pre-replicative complex. One site occurs on the upper strand between nucleotides 3914 and 3915 and the other site is located on the lower strand between nucleotides 3940 and 3941 (see Figure 1C). To prove that the observed cuts map the sites of topo II interaction, DNA extracted from VP16-treated cells was digested with λ-exonuclease and immunoprecipitated with anti-topo II antibody. TD-PCR analysis of the precipitated DNA (see Figure 1B, lanes 4 and 5) using primer set D (that explores the cut in the lower strand) identifies the same stops, in agreement with the property of topo II to be covalently bound to the 5′ end of DNA, thus making it resistant to λ-exonuclease action. Also in the case of topo II (see Figure 1B, lanes 6, 7 and 11, 12), photo-footprinting analysis did not indicate any gross disruption of the origin structure caused by the topo II block, with the exception of the presence of the topo II-mediated cleavage. For both topo I and II, no cleavages were observed in the absence of poison treatment (see Figure 1A, lanes 2 and 11, and Figure 1B, lanes 2 and 9). Thus, both topos interact with the origin area, within the sequence covered by the replicative complexes (Dimitrova et al, 1996; Abdurashidova et al, 1998; Paixao et al, 2004), close to and ahead of the start sites, on the templates for leading strand synthesis. Sequence dependence of the selection for the cleaved bonds We investigated whether the selection of cleaved origin residues by these enzymes is dictated by direct enzyme/DNA sequence recognition or by other factors. When pure topo I was incubated with origin DNA, CPT induced in the lower strand a single cut in exactly the same position as the one observed in vivo (see Figure 2A, lane 3 and WT row of Figure 2C). The same result was obtained with different CPT derivatives (see Figure 2A, lanes 5 and 6). In the case of the upper strand, four topo I-mediated CPT-induced cleavages were observed, of which one coincides with the in vivo cleavage mapped on the same strand (see Figure 2A, lane 10). All the cleavages observed in vitro are topo I-mediated, as incubation of origin DNA with CPT in the absence of topo I showed no cleavages (see Figure 2A, lanes 1 and 8). Figure 2.Interaction of topo I and II with the lamin B2 origin in vitro. (A) Detection of the in vitro topo I cleavages stabilized on the lower strand by CPT (lane 3), 7-[CH2–Tris] CPT (lane 5) or gimatecan (lane 6), and on the upper strand by CPT (lane 10); lanes 7 and 12: Maxam–Gilbert sequencing reactions; the position of the cleavages also present in vivo is indicated by an asterisk. (B) Effect of base substitution mutations in the lamin B2 origin on topo I-mediated cleavage. (C) Sequence of the origin portion covered by the replicative complexes; the position of substituted bases is highlighted; the position of in vitro topo I-cleavable complexes is indicated by filled triangles; the asterisks indicate the position of the topo I cleavages also present in vivo. (D) Detection of the in vitro VP16-induced topo II cleavages introduced by the enzyme alone (lanes 1–5) or by topo II as part of a complex with nuclear proteins (lanes 6–14); lane 9: the origin DNA incubated with the nuclear extract and VP16 was immunopurified using anti-topo II antibody; black vertical bars indicate the region protected in vivo; the arrows indicate the borders of the region protected in vitro by the origin binding proteins (OBP) as determined by λ-exonuclease digestion; lanes 10 and 14: Maxam–Gilbert sequencing reactions. Download figure Download PowerPoint The replacement of a 10 T stretch, shown to spontaneously acquire in vitro unusual, probably triple-stranded, structures (Kusic et al, 2005) and located close to the in vivo topo I site on the upper strand, with a grossly modified sequence, abolished the two nearby in vitro cleavage sites but not the far away ones (see Figure 2B, lanes 4 and 13, and C). The interaction with the lower strand was abrogated instead by replacement of six nucleotides comprising the topo I binding site (see Figure 2B, lane 7, and C). At the same time, this mutation did not affect any of the upper strand cleavage sites (see Figure 2B, lane 10). Therefore, in spite of the lack of sequence similarity between the mapped topo I cleavages, it is obvious that the enzyme has a clear affinity for a given region. Topo II, instead, is not addressed to its specific sites by direct enzyme/sequence recognition: the pure enzyme, incubated with origin DNA in the presence of VP16, introduced cuts without obvious sequence preference (see Figure 2D, lanes 1–5). Considering the precise in vivo localization of topo II on origin DNA in asynchronous cells, we investigated if the topo II–lamin B2 interaction might be influenced by other nuclear proteins. We took advantage of the fact that we could build in vitro a specific multiprotein complex on origin DNA, which has a definite electrophoretic mobility shift (data not shown) and covers the area from nt 3851 to 4007 (as demonstrated by a λ-exonuclease protection assay, see arrows in Figure 2D), similar to the area protected in vivo in the middle of G1 (Abdurashidova et al, 1998). In contrast to the results obtained with pure topo II, the origin DNA incubated in the presence of the nuclear extract exhibited completely different polymerase stops. In the presence of SDS, but regardless of VP16 addition (in good agreement with previous in vitro data showing that denaturation of topo II stabilizes the cleavage complex; Lee and Hsieh, 1992), origin DNA displays, on both upper and lower strands, the same cleavage pattern as the in vivo-mapped topo II cutting sites (see Figure 2D, lanes 6, 7, 11 and 12). These cuts are indeed due to topo II, as the same cleavages were detected in protein–DNA complexes immunoprecipitated with an anti-topo II antibody (Figure 2D, lane 9). In good agreement with the lack of sequence specificity of pure topo II, and the fact that it can only recognize the same in vivo sites as part of a complex with other proteins, replacement of 10 nucleotides near the lower strand topo II cleavage site (nt 3943–3952) did not abolish any of the topo II cuts introduced by the nuclear extract on either strand (data not shown). Thus, in contrast to topo I, topo II has no intrinsic sequence affinity for the lamin B2 origin, but is directed at precise sites by other proteins present in the nucleus. Interaction of topos with the origin along the cell cycle To investigate the cell-cycle-dependent interaction of topo I with the origin, cells were collected in M, early G1, middle G1, late G1 (mimosine block), G1/S border–early S (aphidicolin block) and S phase, and subjected to CPT treatment and LM-PCR analysis using the primer sets shown in Figure 3A. The results in Figure 3B show that topo I is clearly present on the origin, at the already identified sites, in M and early G1, leaves it before the middle of G1 and reappears again at the same sites in late G1–G1/S border, finally to leave the origin again in S, when the lamin B2 origin has fired. The presence of the CPT-induced cuts in mimosine-arrested cells (late G1) shows that the enzyme is acting before synthesis starts, as this drug does not allow entry in S, whereas aphidicolin may not cause an absolute block of initiation. Figure 3.Topo I interacts with the lamin B2 origin in a cell-cycle-dependent manner, and is a member of the origin binding complex. (A) Localization and orientation of the primer sets in the analyzed region; the positions of the detected topo I–DNA complexes are indicated by vertical arrows. (B) LM-PCR-mediated detection of CPT-induced topo I cleavage complexes on the lower and upper strands in different moments of the cell cycle; G, in vitro DMS-treated genomic DNA. (C) Identification of the presence of topo I on the CPT-induced cleavage sites and interaction of the enzyme with Orc2p; in the upper portion are shown the positions of the primers utilized (arrows) relative to topo I cutting sites; HeLa cells subjected to CPT treatment were crosslinked or not with DSP, lysed and the DNA was immunopurified with anti-topo I, anti-Orc2p or unrelated antibodies; the lower portion shows the PCR analysis of untreated genomic DNA (lanes 1 and 17), of the DNA immunopurified with anti-topo I antibodies (lanes 2–4), with anti-Orc2p antibodies (lanes 13–16) or with unrelated antibodies (lanes 5–12). (D) Topo I co-immunoprecipitates with Orc2p in HeLa nuclear extract: Western blot of proteins immunoprecipitated with anti-Orc2p antibody and assayed with anti-topo I or anti-Orc2p antibodies. (E) Formaldehyde crosslinking shows that both topo I and Orc2p associate with the lamin B2 origin in late G1; the DNA from formaldehyde crosslinked HeLa cells was immunopurified using anti-topo I or anti-Orc2 antibodies or pre-immune serum and subjected to competitive PCR analysis; B48, origin region; B13 non-origin region. Download figure Download PowerPoint The cell-cycle dependence of the origin interaction with topo I is also observable at the same sites and at the same cell-cycle moments in human fibroblasts, synchronized in G0 by serum starvation, and in HeLa cells collected in M by nocodazole treatment, released into G1 in the absence of any drug (see Supplementary Figure 2). We extended our analysis of the presence of topo I far from the replicative complex area to a total length of 1500 bp (see Figure 3A for the position and direction of all primer sets used). On the left side, no other cleaved bond was observed on either strand, whereas on the right side only one further site was found to be present in S phase on the lower strand, far from the area covered by the replicative complexes, between nt 4335 and 4340 (primer set B). This corresponds to the region located on the 5′ side of the template of the housekeeping TIMM13 gene, just downstream of the promoter. It is conceivable that the action of topo I at this site is related to transcription. Thus, the in vivo interaction of this enzyme with the origin within the replicative complex area appears to be focused by its intrinsic affinity towards this region in the close neighborhood of the start sites of synthesis. To ascertain whether the CPT-induced cleavages are indeed topo I-mediated, we designed the experiment reported in Figure 3C; the DNA of the cells synchronized in late G1 with mimosine and treated or not treated with CPT was isolated and digested with a restriction enzyme, yielding a fragment encompassing the two sites cut in vivo inside the area covered by the replicative complex. The DNA–protein complexes derived from the CPT-treated or not treated cells were subjected to immunoprecipitation with anti-topo I antibodies; the DNA preparations were subjected to PCR with the primer sets located at the positions shown in Figure 3C. If the cuts were actually caused by topo I, we should find in the precipitate the fragments close to the sites cut in vivo. As expected, in control untreated cells, total DNA gave the three expected PCR products (one encompassing and two not encompassing the cleaved bond) (see Figure 3C, lane 1). In contrast, in the presence of CPT, the anti-topo I antibody selectively immunoprecipitated only the two fragments on either side of the cleaved bonds, in good agreement with the property of topo I to be covalently bound to the 3′ end of the DNA (see Figure 3C, lane 2). Considering the presence of topo I in late G1–G1/S border within the area covered by the replicative complex, it was tempting to assume that topo I acts as part of the origin binding complex. To investigate if this is indeed the case, we treated the cells synchronized in late G1 with both CPT and the protein–protein crosslinking agent dithiobis-(succinidylpropionate) (DSP), together or separately. The DNA–protein complexes were then isolated, immunoprecipitated with anti-Orc2p antibody and analyzed by PCR with the same primers shown in Figure 3C. The results reported in lanes 13–16 of Figure 3C highlight that anti-Orc2p antibody does not precipitate any DNA when CPT and DSP treatments were performed separately (see lanes 13 and 15). In contrast, in the case of the combined treatment, antibody against Orc2p selectively immunoprecipitated exactly the same DNA fragments shown to be covalently bound to topo I (see Figure 3C, lane 14), demonstrating that topo I and Orc2p are interacting either directly or through some intermediate protein, being in any case both members of the complex. The interaction of the two molecules is confirmed by the observation that when a nuclear extract was precipitated with the anti-Orc2p antibody, the precipitated proteins were shown to contain topo I (see Figure 3D). Finally, a chromatin immunoprecipitation assay performed on late G1-arrested cells with either anti-Orc2p or anti-topo I antibody yielded a clear enrichment for the lamin B2 origin sequence (Figure 3E), confirming that both proteins are found on the origin at this moment of the cell cycle. Interestingly, as shown in Figure 3C, both topo I residues located on the lower and upper strands interact with Orc2p, indicating that this ORC member interacts with topo I on the upper and lower strands, although we cannot tell whether the same or two different Orc2p molecule(s) interact(s) with the topo I molecules located on either side of the start site of synthesis. The experiments reported in Figure 3C–E allow us to conclude that topo I is closely associated with the human origin binding complex on lamin B2. In summary, topo I binds selectively the templates of leading strand synthesis, each at one precise bond ahead of and close to the start site of DNA synthesis. The binding occurs in M, early G1 and late G1–G1/S border. At this last moment, topoisomerase I is adjacent to Orc2p. It is tempting to consider the interaction close to the start site (in association with Orc2p) correlated with the topological demands of the origin for its proper and timely function, considering that the origin fires at the onset of S. Analysis of the cell-cycle-dependent behavior of topo II, as reported in Figure 4A–C, shows that the enzyme is bound at the indicated sites in M and in the middle of G1. When we extended our analysis to a total of 1500 bp with different primer sets (see Figure 4B), we identified two stops with the primer set G (Figure 4C). Besides the cleavage located at the origin, we also observed another cleavage, constant throughout the cell cycle and located over 200 bp away from the origin-bound topo II molecule. When with the same primer set G we analyzed DNA immunoprecipitated with anti-Orc2p antibody from the cells collected in the middle of G1 treated with VP16 and DSP together or separately, we detected just one stop corresponding to the origin-bound topo II only when VP16 and DSP were used together, showing that just this topo II molecule interacts with Orc2 (Figure 4D). Therefore, only this topo II molecule is a member of the human origin binding complex, in agreement with the observation that only this site is comprised within the area covered in vivo by the replicative complexes. Figure 4.Topo II interacts with the lamin B2 origin in a cell-cycle-dependent manner, and is a member of the origin binding complex. (A) TD-PCR-mediated detection of VP16-induced topo II cleavages on the upper strand; G, in vitro DMS-treated genomic DNA. (B) Localization and orientation of the primer sets in the region analyzed; the positions of the detected topo II–DNA complexes are indicated by vertical arrows. (C) TD-PCR-mediated detection of VP16-induced topo II cleavages on the lower strand; G, in vitro DMS-treated genomic DNA. (D) TD-PCR analysis of DNA immunopurified with anti-Orc2p (lanes 3–6) or unrelated antibodies (lanes 1 and 2) from HeLa cells synchronized in the middle of G1, treated with VP16, DSP or both; topo II binding sites at or outside of the origin are indicated with one or two asterisks respectively; G, in vitro DMS-treated genomic DNA. Download figure Download PowerPoint Thus, within the 1500-bp explored area, topo II is bound in vivo only to three sites: one site is 170 bp removed to the left of the replicative complex area and could correspond to a scaffold attachment region. The other two sites lie very close to the start sites of bidirectional synthesis. The enzyme is bound to these two sites in mitotic chromosomes, like topo I; preliminary data indicate that these two enzymes are not bound contemporaneously to origin DNA: simultaneous treatment of asynchronous cells with CPT and increasing concentrations of VP16 followed by analysis of the induced cuts using primer set D showed the presence of both the topo I and II stops in the lower strand, an outcome possible only if the enzymes are present in different molecules, as we use poison concentrations giving maximal effect (data not shown). In view of the demonstrated presence of topo I on the origin in early G1, we surmise that topo II binds the origin in early mitosis, possibly contributing to the packing of metaphase chromosomes, and topo I binds towards the end of M. Topo II is then present at the origin in the middle of G1, where it interacts with Orc2 inside the pre-replicative complex, pointing towards a role of topo II in the assembly and reorganization of the G1 pre-replicative complex. In all the experiments described here, we have invariably observed the presence of topo II on one strand only, and could never obtain evidence for the interaction with the complementary strand, 4 bp removed, which is a described property of this homodimeric enzyme. This observation has many precedents, as conditions in which the enzyme remains bound, after poison blockage, to one strand only have been described in several instances (Muller et al, 1988; Lee et al, 1989). Furthermore, one has to consider that the action of topo II on the origin that we investigated invariably occurs in the context of a multiprotein complex, in vivo or in vitro, and that protein–protein interactions may be at the base of the preference for one s
Referência(s)