Kinetoplast DNA Maxicircles: Networks Within Networks (Trypanosomes/Topology) THERESA A

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 7809-7813, August 1993 Biochemistry Kinetoplast DNA maxicircles: Networks within networks (trypanosomes/topology) THERESA A. SHAPIRO Division of Clinical Pharmacology, Department of Medicine, The Johns Hopkins University School of Medicine, 301 Hunterian Building, 725 North Wolfe Street, Baltimore, MD 21205-2185 Communicated by Paul Talalay, May 20, 1993 (receivedfor review, April 27, 1993) ABSTRACT Kinetoplast DNA (kDNA), the mitochondrial trypanosomes, the location of mitochondrial topoisomerase DNA of trypanosomes, is an enormous network of interlocked II with respect to kDNA is unknown. minicircies and maxicircles. We selectively removed miinicir- Early EM studies showed that selective removal of cles from Trypanosoma equiperdum kDNA networks by restric- maxicircles from kDNA networks by restriction enzyme tion enzyme cleavage. Maxicirdes remained in aggregates that digestion left residual catenanes of minicircles (14, 15). were resistant to protease or RNase and contained no residual Treatment with topoisomerase II established unequivocally minicircies, but were resolved into circular monomers by that each kDNA network contained several dozen unit-length topoisomerase II. Maxicirdes thus form independent catenanes circular maxicircles (16). However, the arrangement of within kDNA networks. Heterogeneity in the size, composition, maxicircles within the minicircle scaffold has remained spec- and organization of maxicircle catenanes reflects changes that ulative (10). In nonreplicating networks, maxicircle loops occur during kDNA replication. The rosette-like arrangement protrude from the margins of the matrix. In replicating of maxicircle catenanes is distinctly different from that of networks from trypanosomes (but not from Crithidia), minicircle catenanes. Trypanosome kDNA networks reveal maxicircles are strikingly apparent as a cluster between the unique topological complexity: they are composed of entirely two lobes of the separating daughter networks. dissimilar catenanes that are in turn extensively interlocked T. equiperdum maxicircles are homologous with those of with one another. Trypanosoma brucei (17), but the minicircles are distinctive because they are all of one sequence (18). We identified an Kinetoplastid protozoa are pathogenic to humans, livestock, enzyme that would accomplish the statistically unlikely and commercially important plants. A distinguishing mor- cleavage of minicircle, but not maxicircle, DNA. In this phologic feature of these organisms is their massive mito- paper we describe the maxicircle product produced when Spe chondrial DNA (termed kinetoplast DNA, or kDNA), which I selectively removes all minicircles from T. equiperdum repeatedly proves to be structurally and functionally fasci- kDNA networks. This product is a catenane of interlocked nating. The single mitochondrion of each cell contains one maxicircles. Maxicircles therefore form networks within kDNA network composed of thousands of interlocked min- kDNA networks. icircles and tens of maxicircles (reviewed in refs. 1-3). Depending on the species, minicircles are about 1 kb each and MATERIALS AND METHODS there may be hundreds of different sequence classes in a network; maxicircles are about 20 kb each and of identical Trypanosomes and kDNA Isolation. T. equiperdum trypa- sequence. Maxicircles are analogous to other mitochondrial nosomes (Pasteur Institute strain BoTat 24; African trypa- DNAs in that they code for mitochondrial rRNA and some nosomes closely related to T. brucei) were grown in female mitochondrial proteins. The unique genetic role of minicir- Wistar rats, isolated from blood by means of DEAE- cles has only recently been discovered: they code for guide cellulose, and lysed with SDS. The lysate was treated with RNA molecules that direct the editing of maxicircle tran- RNase and proteinase K, phenol-extracted, and dialyzed scripts to generate fuinctional open reading frames (reviewed (11). Dialysate was layered over sucrose cushions (20% in 10 in refs. 4-6). mM NaCl/100 mM EDTA/10 mM Tris HCl, pH 8.0) and Replication of kDNA entails the production of two daugh- centrifuged (19,000 rpm, 4°C, Beckman SW28 rotor, 1 hr). ter networks identical in size and composition to the parent The pelleted networks were collected, dialyzed, and centri- (reviewed in refs. 7 and 8). Not surprisingly, this process is fuged (12,000 rpm, 4°C, Savant microcentrifuge, 1 hr). highly complex. Covalently closed minicircles are individu- Enzyme Digests. Unless indicated otherwise, digestion was ally decatenated, and they replicate free of the network. for 3 hr at 37°C; buffers provided by the enzyme supplier were Daughter minicircles are segregated from one another and supplemented with 0.5 mM dithiothreitol. DNA was phenol- attached to the periphery ofthe network. Much less is known extracted and ethanol-precipitated between reactions. Net- about maxicircle replication, which involves a rolling-circle works (3 p,g/ml) were decatenated with topoisomerase II mechanism (9). In trypanosomes, the growing network from T4 phage [gift of Ken Kreuzer (19); 2.5 units/ml] for 1 evolves into a dumbbell shape and then divides (10). The hr at 370C in 60 mM KCl/10 mM MgCl2/0.5 mM dithiothrei- numerous topological interconversions necessary to synthe- tol/0.5 mM Na2EDTA, 0.5 mM ATP/40 mM Tris HCl, pH size and remodel kDNA are catalyzed by topoisomerases. A 7.8, with bovine serum albumin (50 ,ug/ml). To analyze for mitochondrial type II DNA topoisomerase was demonstrated residual minicircles, networks (3 p,g/ml) were digested at in Trypanosoma equiperdum by drug inhibition studies (11). 37°C for 3 hr with Spe I (50 units/ml) and for 1.5 hr after Two distinct type II topoisomerases have been isolated from addition ofmore Spe 1 (25 units/ml). An aliquot was taken for Crithidiafasciculata and immunolocalized to the kinetoplast gel analysis and the pH was adjusted to 9.5 with KOH prior (refs. 12 and 13; J. Shlomai, personal communication). In to addition of A exonuclease (33 units/ml). The mixture was incubated at 370C for 1 hr and the reaction was quenched with phenol. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: kDNA, kinetoplast DNA. 7809 Downloaded by guest on September 23, 2021 7810 Biochemistry: Shapiro Proc. Natl. Acad. Sci. USA 90 (1993) Gel Electrophoresis. Maxicircle DNA was fractionated in A B 0.6% agarose gels (18-21 hr; 3 V/cm; 24°C) in 90 mM Tris/92 1 2 3 4 1 2 3 4 mM boric acid/2.5 mM EDTA, pH 8.3, containing ethidium bromide (1 pg/ml). Size markers for maxicircles were Hin- dIII or Bgl II digests of A DNA. Nicked and covalently closed circular maxicircles were identified by repairing decatenated s networks with ligase, DNA polymerase, and dNTPs; or by f -N nicking decatenated networks with DNase I (data not shown). Markers for minicircles were Hae III or Hpa II digests of 4X174 replicative-form DNA. Electrophoresis of 23.1- * -c minicircle DNA and transfer to GeneScreen (NEN) have 0 ?s - L been described (11). 22.0- Probes and Hybridization. Plasmid pTKH128 (gift of Ken 2.7- Stuart; ref. 20) contains a 7-kb HindlIl T. brucei maxicircle fragment inserted into pBR322. The insert was excised with HindIll, purified by gel electrophoresis, and radiolabeled by the random primer method. Preparation of probes from full-length cloned T. equiperdum minicircle DNA was de- 1.7- scribed previously (11). Membranes were prehybridized at 55°C for 30 min in 500 mM sodium phosphate, pH 7.2/10 mM EDTA/1% bovine serum albumin/7% SDS (50 ,l/cm2) and 1.4 then hybridized for 12-18 hr at 55°C in prehybridization solution (25 p1/cm2) containing 10 pmol of 32P-labeled probe 1.1 - * -N (0.6-6 x 106 cpm/pmol). Filters were washed four times (15 min each, 55°C) in 600 mM NaCl/60 mM sodium citrate, pH :4 7.0/0.5% SDS. 0 9.-- EM. DNA was prepared for microscopy by the formamide technique (21). Samples on parlodion-coated copper grids were stained with uranyl acetate and rotary shadowcast with platinum/palladium. Micrographs were taken on a Zeiss *c transmission model EM 1OA electron microscope. DNA contour lengths were determined with a map measurer (mod- FIG. 1. Analysis of Spe I digests. Purified T. equiperdum kDNA el 11-11, Alvin Co., Windsor, CT). networks were digested with enzymes, and the samples were divided and analyzed for minicircle (A) or maxicircle (B) DNA. (A) DNA was electrophoresed in 1.5% agarose containing ethidium bromide, blot- RESULTS ted, and probed with 32P-labeled minicircle DNA. Networks were Spe I Yields Linear Minicircles and Maxicircle Aggregates. treated with no enzyme (lane 1), BamHI (lane 2), topoisomerase II (lane 3), or Spe I (lane 4). Intact networks are too large to enter the To test whether Spe I selectively cleaves minicircles, purified gel and are variably lost from the slot when the gel is processed for kDNA networks were digested with enzyme and the sample transfer. (B) Same reactions as in A, but DNA was electrophoresed was split to analyze minicircle and maxicircle products. The in 0.6% agarose, blotted, and probed with 32P-labeled maxicircle DNA was separated by gel electrophoresis, blotted, and DNA. Each lane contained 15 ng of kDNA. N, circles containing probed with cloned 32P-labeled minicircle or maxicircle DNA nicks or gaps; L, linearized circles; C, covalently closed circles; S, (Fig. 1 A and B, respectively). As predicted from the se- smear of maxicircle DNA. Scales indicate size of marker fragments quence, the minicircle products of Spe I migrated as a single in kilobases; arrows indicate slots. 1-kb band (Fig. 1A, lane 4) that represented full-length linearized molecules, clearly distinct from nicked and cova- kDNA network preparations, Spe I digests, spreads, EM and lently closed circular monomers generated by topoisomerase film processing), 55 structures (29 plus 26) were evaluated.

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