Replicating, Covalently Closed, Circular DNA from Kinetoplasts

Proc. Nat. Acad. Sci. USA Vol. 69, No. 6, pp. 1642-1646, June 1972

Replicating, Covalently Closed, Circular DNA from Kinetoplasts of Trypanosoma cruzi (electron microscopy/dye equilibrium centrifugation/replicating units/trypanocidal drugs/Berenil) CH. BRACK*, E. DELAIN, AND G. RIOUt Laboratory of Molecular Pharmacology and Electron Microscopy, Institut Gustave-Roussy, 94-Villejuif, France Communicated by FranCois Jacob, April 5, 1972

ABSTRACT When Trypanosoma cruzi are treated with DNA Extraction. The kDNA was prepared (9) by extraction Berenil, a trypanocide, their kinetoplast DNA contains an of total cell DNA and fractionation of the kDNA in a increased proportion of double-branched circular mole- Hg++- cules. These replicating molecules have closed-circular Cs2SO4 gradient. The kDNA was centrifuged in an ethidium template strands; their decrease in density when corn- bromide-CsCl gradient in a Spinco 40.3 fixed-angle rotor. plexed by ethidium bromide in a cesium chloride gradient The two characteristic bands fluorescent under UV light were is proportional to the length of the replicated segments. separated by 15 mm after 60 hr of centrifugation at 35,000 rpm Replication seems to be blocked at specific points, which are equidistantly spaced along the circular kinetoplast and 200C. DNA molecules. Analysis of about 800 replicating forms Electron Microscopy of DNA. Purified kDNA was spread by showed that the lengths of the replicated branches are not distributed at random, but into several populations, which the technique of Lang and Mitani (10), with the following correspond to multiples of 15% of the total contour length modifications. 10-15 ,d droplets of the spreading solution were of 0.5 Am. This distribution evokes a discontinuous re- placed on a Teflon plate. They contained (final concentra- plication process. The problem of whether kinetoplast tions): 0.5 pg/ml of DNA, 15 jg/ml of cytochrome c, 0.05 M DNA is synthesized by successive replication units, or whether and how Berenil might induce specific blocking ammonium acetate, and 50% formamide (Fluka puriss. pro of DNA replication, is discussed. analysi). A small beaker containing formaldehyde (37% The kinetoplast is a specialized region of the unique mito- chondrion of Trypanosomatidae, and contains a very large amount of DNA (kDNA). Recent investigations have pointed out the complex organization of the minicircular and "linear" molecules within the kinetoplast (1-4). When trypanosomes 1. divide, both the nucleus and the kinetoplast are duplicated. 10 Active synthesis of nuclear and kDNA has been revealed by C, 0c incorporation of [1H Ithymidine (5-7). However, little is E known about the mechanism of the equal distribution of the 0. enormous mass of kDNA (several thousands of circular c molecules) and the molecular aspects of its replication. m In the kDNA of exponentially growing Trypanosoma cruzi, .00 replicating double-branched circular molecules are very rare. L- 5 When the trypanosomes are treated with Berenil (4,4'- To diazoamino dibenzamidine diaceturate- 4H20), an increased ID proportion of such replicating circles is observed. This E trypanocidal drug interacts preferentially with the kDNA, z blocking its synthesis and inducing the formation of dys- kinetoplastic trypanosomes (8). The relatively large amount of replicating molecules allowed us to analyze some of their 10 20 30 40 50 60? 70 80 90 morphological and physical characteristics. r=Length of replicated segment (/) MATERIAL AND METHODS FIG. 1. Histogram of the percentages of the replicated lengths Trypanosome Culture. Trypanosoma cruzi were cultured and of 183 circular molecules from total kDNA, from one representa- treated for 4 days with 2 Mg/ml of Berenil (8). tive experiment. The length of replicated segments (r) was calculated and expressed as percentage of the average contour length Abbreviation: kDNA, kinetoplast DNA of the whole circle: * Present address: Department of Microbiology. Biozentrum, (a + b)/2 r = X 100 = % of average contour length University of Basel, Klingelbergstrasse 70 CH4056, Basel, (a + b)/2 + c Switzerland. t To whom requests for reprints should be addressed: Laboratoire (a and b are the replicated segments, and c is the unreplicated de Pharmacologie Moldculaire, Institut Gustave Roussy (94), segment). Peaks are clearly visible at r = 15, 30, 45, and 60%. Villejuif, France. Minor peaks above 60% are less evident. 1642 Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Circular Replicating Kinetoplast DNA from T. cruzi 1643

FIG. 2. Double-branched replicating kDNA molecules. Calculation of r: see Fig. 1. r = A and B: 12%; C: 15%7; D and E: 28%o; F: 30%; G: 357%; H: 42%; I and J: 68%c; K and L: 77%a; M: 90%-replicating dimers; N: 27%; 0: 36%- (X 100,000). .NMerck, pro analysi) was placed beside the droplet, and both of replicating forms could be observed: among about 10() wevre covered with a petri dish. After 20-60 min of diffusion} circular molecules, more than 800 double-branched circles, time, the protein film, which contained DNA, was picked up where photographed. Most of the replicating molecules were on carbon-coated copper grids, dried in 95% ethanol, and free, others were attached to small or complex associations of shadowed with platinum while it was rotated (30 mg, 10 cm, circular molecules. The mean contour length of the replicating 60). The formaldehyde vapors denature the cytochrome c molecules was equal to that of nonreplicating circles. The film at the air-solution interface of the droplet. The molecules relative difference between the lengths of the replicated appear to be well extended, and their length is slightly branches was generally less than 2%. The length of the increased as compared to our previous results obtained with replicated part (r) was calculated and expressed as a per- other techniques (9, 11). Micrographs were recorded on 70-mm centage of the average contour length of the whole circles roll film with a Philips EM 300 electron microscope, at a (see Fig. 1). magnification of X 65,000. Measurements were made with a Fig. 1 presents a histogram of the percentages of the map ruler on negatives enlarged 10-fold with a Leitz profile replicated length (r) of 183 circular molecules from the total projector. Accurate calibration is not needed, because all ourt kDNA from one representative experiment, before its frac- data are expressed as relative values. tionation in an dye-CsCl gradient. The histogram shows that: (a) the length distribution of the replicated segments is RESULTS neither random nor gaussian; (b) there are almost no molecules Comparison of total cell DNA of normal and Berenil-treated that are replicated to 50%: i.e., with three branches of equal trypanosomes by analytical ultracentrifugation reveals a length; (c) there are several equidistantly spaced peaks, decrease of the relative amount of kDNA, from 22 to 14% of which correspond to r = 15, 30, 45, and 60%. Some further the total cellular DNA. The buoyant densities of the nuclear minor peaks above 60% are less evident. About 0.1% of the DNA and kDNA (11) were unchanged. circular molecules are monocircular dimers (contour length The kDNA of untreated trypanosomes is composed of 1.0 gm). Two of them were double-branched circles; the complex associations of "linear" and minicircular molecules, lengths of their replicated segments were 27 and 36% of the as well as many free circles (4). The minicireles have a contour dimer length (Fig. 2, N, 0). length of about 0.5 Mm. Among the circular molecules, we On Fig. 2, we have grouped free replicating molecules observed molecules with two branch points and two segments corresponding to the different peaks of the histogram (Fig. 1), of equal length between these points. Such molecules are as well as the two replicating circular dimers. Most of these currently considered to be replicating intermediates. These molecules appear in the open configuration, some of them with replicating molecules are found in a very low proportion- one twist in the unreplicated branch (Fig. 2, B, E). We have only three for thousands of molecules examined during not detected any single-stranded regions associated with the several years. branch points,. When T. cruzi are treated for 4 days with Berenil, the com- After kDNA fractionation in gradient, the 15-mm inter- plex associations of their kDNA molecules are morphologically mediate between the two bands usually observed (covalently- modified (8). In these preparations, an increased proportion closed; nicked and linear molecules) was divided into three Downloaded by guest on October 2, 2021 1644 Biochemistry: Brack et al. Proc. Nat. Acad. Sci. USA 69 (1972)

have been reported from several microorganisms: bacterio- phages-X (13-15) and PM2 (16), animal viruses-SV40 (17-19) and polyoma virus (20-24), bacterial episomes- Col El (25, 26), as well as from the mitochondrial DNA (mtDNA) of higher animals (27, 28). This work shows for the

|" f~~5

1, / lllaf~~2-4

FIG. 3. Three 30%O replicated molecules (I) from fraction f3 FIG. 4A. Centrifuge tube from a Spinco 40.3 fixed-angle rotor. of a gradient (see Fig. 4) Fractions fl and f5 are separated by 1.5 cm. This 1.5-cm zone was (X45,000). separated into three fractions, f2, f3, and f4. equal fractions (f2, f3, and f4), while the lower and upper bands were called fl and f5, respectively (Fig. 4A). Electron microscopic examination of the pooled fractions revealed the presence of replicating molecules in all five fractions. Their relative frequency, however, was different: in f3 and f4, about 2-4% of the free molecules were replicating, in fl, f2, and f5, only 0.5-1%. Fig. 3 shows three replicating molecules, with r = 30%, which are selected in f3. Up to 40 replicating forms can be observed on one square of a 300-mesh grid in these preparations. The histograms of Fig. 4B correspond to the replicating molecules of the five fractions. Fractionfl contains replicating molecules from the first peak (r = 15%). This population disappears progressively in the f2 and f3 fractions, and is absent in fraction f4. Twisted replicating circles can be found in fractions fl and f2. The main peak (r = 30%) reaches its maximum in f3. The replicating circles with r > 50% are mainly found in f3 and f4. Fraction f5 corresponds to the upper band, and contains the nicked replicating forms of all classes. The cumulating histogram (Fig. 4C) of the replicating molecules found in the five fractions shows the same pluri- modal distribution of the replicated lengths (r) as in Fig. 1. Fraction f5 contained rare molecules with one branch point and one or two tails, the lengths of which never exceeded the contour length of the circle. They were considered to be broken replicating molecules, and were not included in the histograms. In some preparations, especially from f2 and f3, replicating molecules have been detected in association with circular molecules. They are connected to other circle(s), either by their unreplicated branch c (Fig. 5A) or by one or both of the replicated segments a and b (Fig. 5B and C). Replicating molecules can also be detected in large associations of circles r = Length of replicated segment (%) (Fig. 5D). FIG. 4B. Histograms of the percentages of the replicated DISCUSSION lengths of the molecules found in fractions fl-f5. FIG. 4C. Cumulative histogram of the total of 374 molecules Double-branched circular DNA molecules, as initially ob- measured from fractions fl-f5. The peak distribution is the same served by Cairns for Escherichia coli chromosomes (12), as for unfractionated kDNA. Downloaded by guest on October 2, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Circular Replicating Kinetoplast DNA from T. cruzi 1645

first time the existence of replicating double-branched circles in the kDNA of trypanosomes. The dye-equilibrium centrifugation with ethidium bromide has shown that these replicating molecules are distributed all along the gradient, and that their position in the gradient depends on the length of the replicated branches. The longer the replicated segments are, the lower the apparent density at which they band in the gradient. These results are in agreement with those of Jaenish et al. (18), Sebring et al. (19), and Bourgaux-Ramoisy (24), which indicate that replicating molecules contain covalently-closed circular template strands. The open or twisted appearance on the micrographs is not necessarily a proof of the nicked or closed configuration of the minicircular kDNA molecules; their small contour length provides them with particular topological constraints. The presence of complex associations of molecules, some of them replicating, suggests, as previously mentioned by .Meinke and Goldstein (23), that replicating events may lead to the formation of different types of polymeric molecules. Similarly "catenations" of molecules have been described in replicating intermediates of Col El (26). Simple interpreta- tions of similar pictures will be presented and discussed separately. In the kDNA of untreated trypanosomes, only very few replicating molecules can be detected. If we consider that the speed of DNA replication of E. coli is 25 sm/min (29) and that of mammalian chromosome 2 gm/min (30), it is possible to estimate the time of replication of the very small kDNA (0.5 ,m) to be only a few seconds. As one cell cycle of T. cruzz m!&1~~~~~~~~ FC takes about 24 hr, and as we do not have synchronized cultures, the probability of extracting the molecules during replication is rather small. FIG. 5. Association of one replicating molecule with un- Berenil treatment of T. cruzi increased the proportion of replicating circular molecule(s). a and b are the replicated seg- replicating molecules in the kDNA by a factor of 103. We ments, c is the unreplicated part. The molecules are attached by have obtained similar results with the related trypanocidal c (A), by a (B) or by a and b (C). A replicating circle is attached by diamidine: hydroxystilbamidine. This apparent accumulation the unreplicated part c to two molecules of a complex association of replicating circles does not seem to be due to a stimulation (D) (X 100,000). of kDNA synthesis, for there are several experimental argu- ments that indicate that Berenil interferes with DNA metabo- four small denaturation loops appear (unpublished results). lism (see ref. 8). The accumulation of replicating forms may Their relative position on the circular molecule corresponds indicate that Berenil does not block the replication process to the relative position of the replication block. Thus, it is at initiation, but at different specific points that are regularly possible that the AT-rich regions that denature first by distributed along the circles, dividing them into segments alkaline treatment are those that preferentially bind Berenil. corresponding to multiples of 15% (i.e., about 0.075 um) of The drug may interfere more-or-less directly with the en- the total contour length (0.50 jm). Unlike kDNA, SV40 (17) zymes of DNA replication, e.g., at the reinitiation sites of the- and polyoma virus DNA (21) contained a majority of mol- replication units. On the other hand, DNA synthesis may ecules in a late stage of replication. Such a distribution be blocked indirectly; if Berenil acts at the level of tran- could be the result of a nonconstant rate of replication. scription, an accumulation of messenger RNA or RNA The display of successive equal steps of replicated lengths is polymerase may stop the replication process. Indeed, it has consistent also with the hypothesis of discontinuous DNA been shown that RNA polymerase of E. coli binds preferen- replication. Okasaki (31) described short DNA pieces syn- tially to the A T-rich regions of bacteriophagef DNA (33). thesized during replication of E. coli chromosomes. "Repli- Kasamatsu et al. (34) and Arnberg et al. (35) have described cation units" have also been discussed by Inman and Schn6s a differentiated zone comprising a single-stranded segment on in connection with the replication forks of X DNA (15). The mtDNA molecules from mouse and chicken cells. Similar 0.075-pm segments present on kDNA are, however, much structures have also been described in mtDNA from human smaller than those reported above. We are not able to deter- blood cells (Riou, G. & Paoletti, C., Eur. J. Biochem., sub- mine whether kDNA naturally contains similar very short mitted). Molecules containing such displacement loops replication units, or whether the short replication steps are (D-loops) were suggested to be intermediates in mtDNA induced by treatment with Berenil. synthesis. Unlike the monocircular dimers of this mtlNA, Berenil binds preferentially to AT-rich regions of DNA which was shown to contain two D-loops 1800 apart from (32). The kDNA of T. cruzi contains 60%0 AT (11). When each other (34), the kDNA dimer molecules seem to replicate kDNA molecules are partially denatured at alkaline pH, as one single unit. Electron microscopy of replicative inter- Downloaded by guest on October 2, 2021 1646 Biochemistry-: Brack et al. Proc. Nat. Acad. Sci. USA 69 (1972)

mediates of T4 DNA has shown the presence of loop struc- 12. Cairns, J. (1963) J. Mol. Biol. 6, 208-213. tures displaying two single-stranded "whiskers" (36). Such 13. Kiger, J. A. & Sinsheimer, R. L. (1971) Proc. Nat. A/cad. Sci. USA 68, 112-115. single-stranded zones could not be detected on the kDNA 14. Ogawa, T., Tomizawa, J. I. & Fuke, M. (1960) Proc. Nat. molecules. Polyoma virus replicating DNA molecules contain Acad. Sci. USA 60, 861-865. less than 2% single-stranded material (24). If kDNA circles 15. Inman, R. B. & Schn6s, M. (1971) J. Mol. Biol. 56, 319- contained an equal proportion of single-stranded DNA, 325. 16. Espejo, R. T., Canelo, E. S. R. & Sinsheimer, R. L. (1971) these regions would correspond to pieces of only 100 A that J. Mol. Biol. 56, 597-621. would be undetectable with the present electron microscope 17. Levine, A. J., Kang, H. S. & Billheimer, F. E. (1970) J. Mol. techniques. Biol. 50, 549-568. As no genetic markers are known on the kDNA circles, it is 18. Jaenisch, R., Mayer, A. & Levine, A. (1971) Nature New not possible to localize the origin of replication. Further Biol. 233, 72-75. 19. Sebring, E. D., Kelly, J. J., Jr., Thoren, M. M. & Salzman, denaturation studies should give us a physical map of the N. P. (1971) J. Virol. 8, 478-490. replicating kDNA, which will enable us to determine the 20. Hirt, B. (1969) J. Mol. Biol. 40, 141-144. starting point and direction of its replication. 21. Bourgaux, P., Bourgaux-Ramoisy, D. & Seiler, P. (1971) Many important questions concerning kD)NA remain J. Mol. Biol. 59, 195-206. 22. Bourgaux, P. & Bourgaux-Ramoisy, D. (1971) J. Mol. unanswered. Nothing is known about the genetic content of Biol. 62, .513-524. kDNA or the significance of its very high concentration in the 23. Meinke, W. & Goldstein, 1). A. (1971) J. Mol. Biol. 61, kinetoplast. Is it a repetitive DNA or a genetic amplification 543-563. of one or a few genes? This small DNA cannot code for many 24. Bourgaux-Ramoisy, 1). (1971) Biochem. Biophys. A cta proteins. Our results show, however, that each of the several 254, 412-414. 25. Inselburg, J. & Fuke, M. (1970) Science 169, 590-592. thousands of minicircles is able to replicate independently. 26. Fuke, M. & Inselburg, J. (1972) Proc. Nat. Acad. Sci. USA This research was supported by a grant of the "Recherche 69, 89-92. Miedicale 27. Kirschner, R., Wolstenholme, 1). R. & Gross, N. J. (1968) Franqaise." Proc. Nat. A cad. Sci. USA 60, 1466-1472. 1. Simpson, L. & )a Silva, A. (1971) J. Mol. Biol. 56, 443- 28. Paoletti, C., Riou, G. & Pairault, J. (1972) Proc. Nat. Acad. 473. Sci. USA 69, 847-850. 2. Renger, H. & Wolstenholme, D. R. (1971) J. Cell Biol. 29. Helmestetter, C., Cooper, S., Pierucci, 0. & Revelas, E. 50, 533-540. (1968) Cold Spring Harbor Symp. Quant. Biol. 33, 809- 3. Laurent, M. & Steinert, AI. (1970) Prcc. Nat. Acad. Sci. 822. USA 66, 419-429. 30. Huberman, J. A. & Riggs, A. 1). (1968) J. Mol. Biol. 32, 4. Delain, E., Brack, Ch., Lacome, A. & Riou, G. (1972) in 327-341. Symposium on the Comparative Biochemistry of Parasites, 31. Okasaki, 11., Okazaki, T., Sakabe, K., Sugimoto, K., Kain- ed. Van den Bossche, H. (Academic Press, New York), pp uma, R., Sugino, A. & Iwatsuki, N. (1968) Cold Spring 167-184. Harbor Symp. Quant. Biol. 33, 129-143. 5. Anderson, W. & Hill, G. C. (1969) J. Cell Sci. 4, 611-620. 32. Festy, B., Lallemant, A. M., Riou, G., Brack, Ch. & Delain, 6. Burton, P. R. & D)usanic, D. G. (1968) J. Cell Biol. 39, E. (1970) C. R. Acad. Sci. Ser. D 271 684-687. 318-331. 33. Shishido, K. & Ikeda, Y. (1971) Biochem. Biophys. Res. 7. Riou, G. (1970) Biochem. Pharmacol. 19, 1524-1526. Commun. 44, 1420-1428. 8. Brack, Ch., Delain, E., Ritou, G. & Festy, B. (1972) J. 34. Kasamatsu, H., Robberson, D. L. & Vinograd, J. (1971) Ultrastruct. Res., in press. Proc. Nat. Acad. Sci. USA 68, 2252-2257. 9. Riou, G. & Delain, E. (1969) Proc. Nat. Acad. Sci. USA 62, 35. Arnberg, A., Van Bruggen, E. F. J., Ter Schegget, J. & 210-217. Borst, P. (1971) Biochim. Biophys. Acta 246, 353-357. 10. Lang, D. & Mitani, M. (1970) Biopolymers 9, 373-379. 36. D)elius, H., Howe, C. & Kozinski, A. W. (1971) Proc. Nat 11. Riou, G. & Paoletti, C. (1967) J. Mol. Biol. 28, 377-382. Acad. Sci. USA 68, 3049-3053. Downloaded by guest on October 2, 2021