The Complex of the 32 kD Endonuclease and 65 kD Protein from Plant Nuclear Matrix Preferentially Recognizes the Plasmid Containing SAR DNA Element

J. Plant Physiol. Vol.

144. pp. 479-484 (1994)

The Complex of the 32 kD Endonuclease and 65 kD Protein from Plant Nuclear Matrix Preferentially Recognizes the Plasmid Containing SAR DNA Element R YSZARD RZEPECKI

Institute of Biochemistry, Wrodaw University, Przybyszewskiego 63/67, 51-148 Wrodaw, Poland Received September 5,1993 . Accepted April 11, 1994

Summary

The preference in recognizing and digesting the plasmid DNA containing SAR DNA region from the human /3-interferon gene was examined. It was demonstrated that when purified to an electrophoretic homogeneity, the 32 kD endonuclease-6S kD protein complex from nuclei of White bush (Cucurbita pepo var. patissonina) is able to preferentially recognize and digest plasmid DNA containing the SAR DNA sequence element. This paper also demonstrates that the same recognition ability revealed intact nuclear matrix preparations. A possible role for the complex in DNA attaching to the nuclear matrix is considered.

Key words: Cucurbita pepo var. patissonina, endonuclease, nuclear matrix, SAR DNA. Introduction

There is no doubt that the chromatin of plant eucaryotic cell nuclei should be highly ordered and compact to make fundamental processes such as replication and transcription possible. Recent data (Wanner et aI., 1991) indicated that plant chromosome structure is consistent with organization of DNA into 10 nm fibers, which are then wound into 30 nm «solenoid" and then into the chromatin loops maintained by the attachment of DNA to a proteinaceous chromosome scaffold (or nuclear matrix). Chromosomal scaffolds andlor nuclear matrices have been isolated from a wide variety of non-plant sources (for review see: van der Welden and Wanka, 1987; Verheijen et aI., 1988). Recent data concerning isolation of nuclear scaffolds or nuclear matrices from plants (Rzepecki et aI., 1989, 1992; Breyne et aI., 1992; Slatter et aI., 1991) indicated morphological similarities to those of other Eucaryotes. Also, the DNA fragment that seems to be responsible for binding the DNA loops to the nuclear matrix (nuclear scaffold) and called SAR or MAR DNA (scaffold or matrix attachment region) revealed the same structure and properties. All now known SAR DNA elements revealed a high AT base pair content (:> 70 %), topoisomerase II consensus sequence (Gasser and Laemmli, 1994 by Gustav Fischer Verlag, Stuttgart

1986 a), and usually sequences related to autonomously replicating sequences (ARSs) from yeast (Sykes et aI., 1988; Amati et aI., 1990), as well as sequences related to T-box and A-box (Gasser and Laemmli, 1986 b; Slatter et aI., 1991). SAR DNA elements play an important role not only in the spatial organization of DNA but also in regulation of transcription (Cockerill and Garrard, 1986; Klehr et aI., 1991) and in normalization of gene expression (Phi Van et aI., 1990; Breyne et aI., 1992). Recent publications demonstrated identification and purification of proteins from nuclear matrices (scaffolds) that specifically bind SAR DNA elements. Romig et a1. (1992) purified to homogeneity a SAF-A protein with an apparent molecular weight of 120 kD from HeLa cells, which have an affinity to several homologous and heterologous SAR elements. Hakes and Berezney (1991) isolated a eDNA clone for matrin FIG, the protein with two putative zinc finger motifs. Von Kries et a1. (1991) purified to homogeneity a 9S kD protein called ARBP from chicken, which specifically binds to MARs from chicken, Drosophila, mouse and human. The most recent paper demonstrates SAR DNA recognition and binding by rat liver lamin B1 (Luderus et aI., 1992) and SATB-1 protein in human thymus cells (Dickinson et aI., 1992). Data provides in our laboratory indicated that the endonuclease is associated with

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nuclear matrix and that the enzyme activity is affected by a polypeptide with an apparent molecular weight of 65 kD from nuclear matrix. Endonuclease associated with nuclear matrix displays highly specific endonuclease activity, whereas when purified to homogeneity it loses its specificity. We isolated and purified to homogeneity both the 32 kD endonuclease and its complex with the 65 kD protein from nuclei of White bush cells. We demonstrated that this 65 kD protein is responsible for specific endonuclease activity of the nuclear matrix associated endonuclease (Rzepecki et aI., 1992). Out most recent results indicated that IgG antiendonuclease inhibits total DNA synthesis in isolated cell nuclei by 70 % as well as replication of nuclear matrix DNA (Rzepecki and Szopa, 1993 sent for publication). In the present study we demonstrate that the isolated complex of 32 kD endonuclease and 65 kD protein is able to preferentially recognize the SAR DNA regions. We also demonstrate that the same properties revealed endonuclease associated with nuclear matrix.

SDS polyacrylamide gel electrophoresis with immobilized DNA Preparation of the gel is the same as in the case of SDS gels with one exception, that before polymerization of the resolving gel, denatured calf thymus DNA was added to an end concentration of 20 Ilg/ mL. After electrophoresis, the gel was washed 4-times for 20 min with 50 mM Tris-Cl buffer, pH 7.0, then twice with 50 mM Tris-Cl containing 2.5 mM CaC\z and 2.5 mM MgC\z followed by incubation for 20 h at 24°C with another portion of the buffer. Then the gel was stained with ethidium bromide and photographed under UV light. Polypeptides possessing nuclease activity after this procedure are revealed by dark bands due to digestion and washing off of DNA in this place.

Endonuclease activity assay The endonuclease activity was determined as described previously (Rzepecki et al., 1989). Plasmid pBR322, pCL and pTZ DNA were used as a substrate in a reaction mixture containing: 50mM Tris-Cl, pH 7.0, 5mM MgCI 2, O.lmM EDTA, 0.2mM DTT, 0.1 mM PMSF and 10 % glycerol. The reaction were carried out at 30°C. The cleavage products were subsequently resolved on 0.8 % agarose gels. Following electrophoresis gels were stained with ethidium bromide and photographed under UV light.

Materials and Methods

Plant material White bush seeds (Cucurbita pepo var. patissonina) were surface sterilized in 1 % H 20 2 , soaked in water for 1 h, sown in a moist germinating-bed and then allowed to germinate for 5 -7 days in the dark. One-three centimeter seedlings were harvested into liquid nitrogen and used immediately.

Isolation of cell nuclei Purification of the cell nuclei was performed as described previously (Rzepecki et al., 1989). All operations were performed at temperatures between 0 and 5 0c.

Preparation of nuclear matrices Nuclear matrices were prepared as described previously (Rzepecki et al., 1989), involving treatment with 2 M NaCl and Triton X-100 with or without O.5mM CUS04 stabilization for 20min at 30°C. The resulting nuclear matrix structures showed a spheroidal shape in phase contrast microscopy.

Purification of complex between 32 kD endonuclease and 65 kD protein Isolation and purification of the endonuclease-65 kD protein complex was performed as described previously (Rzepecki et aI., 1992).

Electrophoresis and immunoblottings Polypeptides were separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis in 15 % gels or in gradient (6 %20%) gels. Proteins were visualized by Coomassie or silver staining. Transfer of the proteins onto nitrocellulose filters and formation of the immune complexes were the same as described previously (Rzepecki et aI., 1989).

pCL and p 12 plasmids pCL and pTZ plasmids were kindly provided by Prof. Jiirgen Bode (GBF, Braunschweig, Germany). Plasmid pCL is the same as pTZ but also contains the 800 bp insert of the SAR DNA flanking human iJ-interferon gene. The SAR character is mainly due to the AATATATTT-tract, which is positioned in an appropriate environment for strand separation (Bode et aI., 1992).

Results

We reported previously that the 32 kD endonuclease reveals its specificity towards plasmid DNA when it is associated either with the 65 kD protein or with nuclear matrix (Rzepecki et al., 1992). The complex of 32kD endonuclease and 65 kD protein was observed mainly in nuclear matrix, and thus it was of interest to investigate whether this complex is able to preferentially recognize and digest SAR DNA elements. To resolve this problem, we purified both the 32 kD endonuclease and the complex nuclease-65 kD protein from cell nuclei by the method described previously (Rzepecki et aI., 1992). We analyzed the isolated material in polyacrylamide gradient (6 % - 20 %) gel electrophoresis under denaturing conditions followed by silver staining. As shown in Fig. 1, the endonuclease and its complex with the 65 kD protein were purified to electrophoretic homogeneity. To ascertain if the 32 kD protein band is really the endonuclease itself we immunologically analyzed both preparations on Western blots. The proteins were stained with IgG anti-endonuclease and second IgG conjugated with peroxidase. As a result we found that the 32 kD protein band is recognized by IgG anti-endonuclease, which suggests that the purified protein is a 32 kD endonuclease. To demonstrate more precisely that in our preparation only the 32 kD protein possesses endonucleolytic activity we resolved it on SDS polyacrylamide gel electrophoresis with immobilized denatured calf thymus DNA. Figure 2 A demonstrates that either in the case of a

The complex of the 32 kD endonuclease and 65 kD protein from nuclear matrix

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Fig. 1: SDS polyacrylamide gradient (6%-20%) gel electrophoresis of isolated 32 kD endonuclease and complex between endonuclease and 65 kD protein. Following electrophoresis, the gel was stained with silver. Lane 1, molecular weight markers (97 kD, 67 kD, 43 kD, 32 kD, 24 kD and 14 kD). Lane 2, isolated complex between 32 kD endonuclease and 65 kD protein (0.5 flg of protein). Lane 3, isolated 32 kD endonuclease (1.2 flg of protein).

complex between the 32 kD protein and the 65 kD protein (Fig. 2 A lane 2) or the 32 kD protein itself (Fig. 2 A lane 3), solely the 32 kD protein showed nuclease activity. The nuclease band corresponds to the protein band recognized by IgG anti-endonuclease (not shown). Figure 2 B demonstrates the endonucleolytic activity of the complex between the 32 kD endonuclease and the 65 kD protein (Fig. 2 Blane 2) and isolated nuclear matrix (Fig. 2 Blane 3) using plasmid pBR322 DNA as a substrate. It is clear that isolated nuclear matrix as well as the complex between 32 kD endonuclease and 65 kD protein revealed highly specific endonucleolytic activity, converting the superhelical plasmid DNA form into the relaxed open circular form. This result supports our earlier finding (Rzepecki et al., 1992) that the endonuclease specificity of the endonuclease-65 kD protein complex is similar to that revealed by intact nuclear matrix. To ascertain whether nuclear matrix associated endonuclease or endonuclease in complex with the 65 kD protein are able to preferentially recognize and digest the SAR DNA element, we used pCL plasmid DNA containing the 800 bp insert of SAR DNA from the human (J-interferon gene target (Bode et al., 1992). We analyzed time dependence of the generation of the OC DNA form and further degradation of the plasmid containing SAR DNA sequence. As a control, pTZ plasmid DNA without this sequence was used. We incubated pCL plasmid DNA (1.5IJ,g) or pTZ (1.3lJ,g) with 0.51J,g of the endonuclease-65 kD protein complex at 30°C for different times and resolved reaction products onto 0.8 % agarose gels in TPE buffer. Figure 3 showed that peL DNA was completely converted into the OC DNA form after 5 min of incubation (Fig. 3, Panel A, lane 2) whereas the su-

Fig. 2: Panel A. SDS polyacrylamide gel electrophoresis, with immobilized DNA polymerized into the gel, of isolated complex: 32 kD endonuclease-65 kD protein and pure 32 kD endonuclease. After electrophoresis, the gel was washed 4-times for 20 min with buffer without magnesium and calcium and then 3-times for 10 min with buffer containing 25 mM Tris HCl, pH 7.0, 2.5 mM MgCl 2 and 2.5 mM CaCb. After 20 h incubation with another portion of the above buffer at 24°C, the gel was stained with ethidium bromide and photographed under UV light. Lane 1, DN-ase I as a molecular weight standard (1.5 flg of protein). Lane 2, isolated 32 kD endonuclease-65 kD protein complex (4.5 flg of protein). Lane 3, isolated pure 32 kD endonuclease (2.5 flg of protein). Panel B. Agarose gel electrophoresis of the supercoiled form of DNA pBR 322 upon treatment with endonuclease-65 kD protein complex and with isolated nuclear matrix. The supercoiled DNA pBR 322 (1.0 flg) was incubated with endonuclease-65 kD protein complex (0.5 flg of protein) (lane 2), and nuclear matrix proteins (60 flg of protein containing 0.3 flg of endonuclease) (lane 3). Lane 3, supercoiled pBR 322 DNA. Samples were incubated at 30°C for 30 min and resolved on 0.8 % agarose gels. Upon electrophoresis, the gel was stained with ethidium bromide and photographed under UV light.

perhelical form of the pTZ DNA was still visible after 5 min of incubation (Fig. 3, Panel B, lanes 2 and 3). Further cleavage of the OC form is faster in the case of pCL DNA (Panel A, lanes 7, 8, 9). The cleavage is completed after 35 to 40 min or 60 min incubation, for pCL and pTZ DNA, respectively. The experiment demonstrates the preference of the endonuclease-65 kD protein complex in recognizing and nicking pCL DNA, which contains the 800 bp SAR element. The next question was whether the intact nuclear matrix also cleaved both plasmid DNAs in the same manner as was observed for the nuclease-65 kD protein complex. To clarify this, nuclear matrices, (60 Ilg protein) containing about 0.3 IJ,g of endonuclease were incubated with 1.5 Ilg of pCL or 1.31J,g of pTZ DNA at 30°C for different times. Products of the reaction were resolved on 0.8 % agarose gels in TPE buffer. Figure 4 demonstrates that incubation of pCL DNA with nuclar matrix for 10 min led to complete conversion of DNA to the OC form (Fig. 4, Panel A, lanes 2 and 3) whereas the superhelical form of DNA is visible even after

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Fig. 3: Agarose gel electrophoresis of the supercoiled forms of pCl (Panel A) and pTZ (Panel B) plasmid DNAs upon treatment with 32 kD endonuclease-65 kD protein complex. Panel A: The supercoiled pCl DNA (1.51tg) was incubated with complex: endonuclease-65 kD protein (O.5ltg of protein) at 30°C for the indicated time. After incubation, DNA samples were resolved onto 0.8 % agarose gels, stained with ethidium bromide and photographed under UV light. lane 1, pCl DNA alone. lnaes: 2-9, pCl DNA after incubation with endonuclease-65 kD protein complex for: lane 2, 5 min; lane 3, 10 min; lane 4, 15 min; lane 5, 20 min; lane 6, 25 min; lane 7, 30 min; lane 8, 35 min; and lane 9, 40 min, respectively. Panel B: The supercoiled pTZ DNA (1.0 Itg) was incubated with endonuclease-65 kD protein complex (O.5ltg of protein) and processed as described above. lane 1, pTZ DNA alone. lanes: 2-8, pTZ DNA after treatment with endonuclease-65 kD protein complex for: lane 2, 5 min; lane 3, 10 min; lane 4, 20 min; lane 5, 30 min; lane 6, 40 min; lane 7, 50 min; and lane 8, 60 min, respectively.

20 min incubation of pTZ DNA (Fig. 4, Panel B, lanes 3 and 4). Further degradation of the pCL OC form is faster when compared with pTZ DNA (Panel A, lanes 5, 6, 7). After 20 min incubation the OC form is not visible in core pCL DNA while in pTZ DNA it exists even after 60 min incubation (Panel B, lanes from 5 to 8). The results of this experiment again indicate the preference in recognition and cleavage of the plasmid DNA containing the 800 bp SAR insert. It could also be suggested that endonuclease in nuclear matrix operates in complex with the 65 kD protein. The above experiments (on recognition and digestion of plasmid DNAs) were done with the superhelical DNA form as substrate. To exclude the possibility of the influence of high ordered conformation of plasmid DNAs on recognition and digestion, the following experiment was conducted. The linear form of pCL (1.0 Ilg) or pTZ (0.9Ilg) plasmid DNA was incubated with O.5llg of the endonuclease-65 kD protein at 30°C for different times and the reaction products were resolved onto 0.8 % agarose gels in TPE buffer. Figure 5 demonstrated that the linear form of pCL plasmid DNA (lanes 1-3) is completely degraded after 10 and 20 min

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Fig. 4: Agarose gel electrophoresis of the supercoiled form of pCl (Panel A) and pTZ (Panel B) plasmid DNA upon treatment with isolated nuclear matrix. Panel A: The supercoiled pCl DNA (1.51tg) was incubated with isolated nuclear matrix (60 Itg of protein, which corresponds to 0.3 Itg of the endonuclease associated with nuclear matrix) at 30°C for the indicated time. After incubation, DNA samples were resolved onto 0.8 % agarose gels, stained with ethidium bromide and photographed under UV light. lane 1, pCl DNA alone. lanes 2-9, pCl DNA after incubation with nuclear matrix for: lane 2, 5 min; lane 3, 10 min; lane 4, 15 min; lane 5, 20 min; lane 6, 25 min; lane 7, 30 min; and lane 8, 35 min. Panel B: The supercoiled pTZ DNA (1.0 Itg) was incubated with nuclear matrix (amount of protein the same as above) and processed as described above. lane 1, pTZ DNA alone. lanes 2-8, pTZ DNA after treatment with nuclear matrix for: lane 2, 5 min; lane 3, 10 min; lane 4, 20min; lane 5, 30min; lane 6, 40 min; lane 7, 50min; and lane 8, 60 min, respectively.

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Fig. 5: Agarose gel electrophoresis of linear forms of pCl and pTZ plasmid DNA upon treatment with 32 kD endonuclease 65 kD protein complex. The endonuclease.-65 kD protein complex (0.5 Itg of protein) was incubated with 1.5 Itg of linear pCl DNA (lanes 2 and 3) or with the linear form (1.0 Itg) of pTZ DNA (lanes 5 and 6) at 30°C for 10 min and 20 min, respectively. lane 1, linear form of pCl DNA alone. lane 4, linear form of pTZ DNA alone. of incubation (lanes 2 and 3, respectively), while the linear form of pTZ plasmid (lanes 4-6) is undegraded after 10min

The complex of the 32 kD endonuclease and 65 kD protein from nuclear matrix

of incubation (lane 5) and only little degraded after 20 min of incubation (lane 6). These results indicate that a high ordered structure of the plasmid DNA has no effect on recognition and digestion of DNA containing the SAR DNA element. This also supports our finding that the endonuclease-65 kD protein complex is able to preferentially recognize and digest plasmid containing SAR DNA sequence elements.

Discussion

We previously reported the presence of the nuclease-inhibitor complex in the nuclei of White bush seeds. The recent data showed that the inhibitor is immunologically related to actin (Szopa and Fahrni, unpublished). It was shown that in phytohormone treated cells endonuclease-inhibitor complex dissociates and 32 kD nuclease binds to nuclear matrix via the 65kD protein (Wisniowska et aI., 1987; Rudnicki et aI., 1988; Rzepecki et aI., 1989). The endonuclease associated with nuclear matrix acquired highly specific activity toward plasmid DNAs, introducing a single nick into one DNA strand, converting the superhelical form (CCC form) of plasmid DNA into the relaxed form (OC form). Recently, we purified to electrophoretic homogeneity the complex of 32 kD endonuclease with 65 kD protein and the endonuclease itself. We demonstrated that this high specificity of endonucleolytic activity of nuclear matrix is due to the association of 32 kD endonuclease with 65 kD protein (Rzepecki et aI., 1992). The present study demonstrated that nuclear matrix or a complex between 32 kD endonuclease and 65 kD protein revealed preference in recognition and cleavage of DNA that contains the SAR element. It should be pointed out that nuclear matrix binds the SAR DNA from /3-interferon with high efficiency in exogenous SAR binding assays, and in gel mobility shift assays (Rzepecki et al. manuscript in preparation). The question arises whether 65 kD protein and 32 kD endonuclease or both proteins are degradation (cleavage) products of another larger protein, e.g. topoisomerase II, which has been found in a great number of copies in nuclear matrices. This possibility was rejected because polyclonal IgG anti-topoisomerase II from cauliflower (kindly provided by Prof. O. Westergaard) does not recognize either 65 kD protein or 32 kD endonuclease, but another protein with an apparent molecular weight of about 140 kD (data not shown). Thus, the demonstrated preference in recognition and digestion of the plasmid containing SAR DNA element (pCL plasmid) is a property of the 32 kD endonuclease sociated with nuclear matrix via the 65 kD protein. Acknowledgement

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BODE, J., Y. KOHWI, L. DICKINSON, T. JOH, D. KLEHR, C. MIELKE, and T. KOHWI-SHIGEMATSU: Biological significance of unwinding capability of nuclear matrix-associating DNAs. Science, 255, 195-197 (1992). BREYNE, P., M. VAN MONTAGUE, A. DEPICKER, and G. GHEYSEN: Characterization of a plant scaffold attachment region in DNA fragment that normalizes transgene expression in Tobacco. The Plant Cell., 4, 463-471 (1992). COCKERILL P. N. and W. T. GARRARD: Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in the region containing topoisomerase II sites. Cell., 44, 273282 (1986). DICKINSON, L. A., T. JOH, J. KOHWI, and T. KOHWI-SHIGEMATSU: A tissue specific MAR/SAR DNA-binding protein with unusual binding site recognition. Cell, 631- 645 (1992). GASSER, S. M. and U. K. LAEMMLI: The organization of chromatin loops: Characterization of a scaffold attachment site. EMBO J., 5,511-518 (1986 a). -

- Cohabitation of scaffold binding regions with upstream/ enhancer elements of three developmentally regulated genes of D. melanogaster. Cell, 46,521-530 (1986 b).

HAKES, D. J. and R. BEREZNEY: Molecular cloning of matrin F/G: a DNA binding protein of the nuclear matrix that contains putative zinc finger motifs. Proc. Natl. Acad. Sci. USA, 88, 61866190 (1991). KLEHR, D., K. MAASS, and J. BODE: Scaffold-attached regions from the human interferon (3 domain can be used to enhance the stable expression of genes under the control of various promoters. Biochemistry, 30, 1264-1270 (1991). LUDERUS, M. E., A. DE GRAAF, E. MATTIA, J. L. DEN BLAAUWEN, M. A. GRANDE, L. DE JONG, and R. VAN DRIEL: Binding of Matrix Attachment Regions to Lamin B1. Cell, 70, 948-959 (1992). PHI-VAN L., J. P. VON KRIES, W. OSTERTAG, and W. H. STRATLING: The chicken lysozyme 5 matrix attachment region increases transcription from heterologous promoter in heterologous cells and dampens position effect on the expression of transfected genes. Mol. Cell. BioI., 10, 2302-2307 (1990). ROMIG, H., F. O. FACKELMAYER, A. RENZ, U. RAMSPERGER, and A. RICHTER: Characterisation of SAF-A, a novel nuclear DNA binding protein from HeLa cells with high affinity for nuclear matrix/scaffold attachment DNA elements. EMBO J., 11, 3431- 3440 (1992). RUDNICKI, K., R. RZEPECKI, and J. SZOPA: Rearrangement of the nuclease-inhibitor complex components within cells of white bush treated with phytohormone. J. Plant Physiol., 132, 658-663 (1988). RZEPECKI, R., G. BULAJ, and J. SZOPA: Endonuclease tightly associated with plant nuclear matrix affected DNA synthesis in vitro. J. Plant Physiol., 134, 364-369 (1989).

RZEPECKI, R., R. SZMIDZINSKI, J. BODE, and J. SZOPA: The 65 kD protein affected endonuclease tightly associated with plant nuclear matrix. J. Plant Physiol., 139, 284 - 288 (1992).

This work was supported by Grant No.4 0088 91 01 from KBN.

SLATTER, R. E., P. DUPREE, and J. C. GRAY: A scaffold-associated DNA region is located downstream of the pea plastocyanin gene. The Plant Cell, 3, 1239-1250 (1991).

References

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VAN DER VELDEN, H. M. W. and F. WANKA: The nuclear matrix Its role in the spatial organization and replication of eucaryotic DNA. Mol. BioI. Reports, 12, 69-77 (1987).

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