Replication of Kinetoplast DNA: an Update for the New Millennium

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International Journal for Parasitology 31 (2001) 453±458 www.parasitology-online.com Invited Review Replication of kinetoplast DNA: an update for the new millennium

James C. Morris*, Mark E. Drew, Michele M. Klingbeil, Shawn A. Motyka, Tina T. Saxowsky, Zefeng Wang, Paul T. Englund

Department of Biological Chemistry, Johns Hopkins Medical School, Baltimore, MD 21205, USA Received 2 October 2000; received in revised form 11 December 2000; accepted 11 December 2000

Abstract In this review we will describe the replication of kinetoplast DNA, a subject that our lab has studied for many years. Our knowledge of kinetoplast DNA replication has depended mostly upon the investigation of the biochemical properties and intramitochondrial localisation of replication proteins and enzymes as well as a study of the structure and dynamics of kinetoplast DNA replication intermediates. We will ®rst review the properties of the characterised kinetoplast DNA replication proteins and then describe our current model for kinetoplast DNA replication. q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Kinetoplast DNA; Trypanosoma; DNA replication

1. Introduction Most studies of kDNA replication in our laboratory, the Ray laboratory (UCLA) and the Shlomai laboratory Protozoan parasites in the family Trypanosomatidae are (Hebrew University) have focused on the insect parasite early diverging eukaryotes that cause important tropical Crithidia fasciculata. Crithidia fasciculata kDNA networks diseases including African sleeping sickness, leishmaniasis, puri®ed from non-replicating cells are remarkably homoge- and Chagas' disease in humans as well as nagana in African neous in size and shape, being planar, elliptically-shaped livestock. All of the trypanosomatid parasites have a structures about 10 by 15 mm in size (see EM in Fig. 1 remarkable mitochondrial DNA, termed kinetoplast DNA showing a segment of an isolated kDNA network). All of (kDNA), that has a structure unlike that of any other the minicircles are covalently closed, relaxed, and linked to known DNA in nature. Within the matrix of each cell's an average of three neighbouring minicircles by single inter- single mitochondrion the kDNA is a network of a few thou- locks (Rauch et al., 1993; Chen et al., 1995). Topologically, sand topologically interlocked DNA circles. There are two the network has a striking resemblance to the chain mail of types of circles, maxicircles and minicircles. Each network medieval armour. Within the parasite's single mitochon- contains several dozen maxicircles (in most species they drion, the network is condensed in a highly ordered fashion range in size from about 20 to 40 kb) and several thousand into a disk-shaped structure about 1 mm in diameter and minicircles (usually 0.5±2.5 kb, although in some species 0.35 mm thick. (Fig. 2 illustrates how the kDNA is they are larger). For a more comprehensive review on condensed into a disk.) The kDNA disk is always positioned kDNA see Shapiro and Englund (1995). Like mitochondrial near the basal body of the ¯agellum and perpendicular to the DNAs from mammalian cells or yeast, maxicircles encode axis of the ¯agellum. Remarkably, there is evidence for a ribosomal RNAs and some of the proteins required for mito- direct physical linkage between the basal body and the chondrial bioenergetic processes. Some RNA transcripts of kDNA network, even though these two structures are sepa- maxicircles are post-transcriptionally modi®ed by the inser- rated by the double membrane of the mitochondrion (Robin- tion or deletion of uridine residues to form functional open son and Gull, 1991). reading frames, a process termed RNA editing. Editing In this review we describe the replication of kDNA, a speci®city is directed by guide RNAs that are encoded by subject that our lab has studied for many years. Our knowl- the minicircles. For a review on editing see Estevez and edge of kDNA replication has depended mostly upon the Simpson (1999). investigation of the biochemical properties and intramitochondrial localisation of replication proteins and enzymes * Corresponding author. Tel.: 11-410-955-3458; fax: 11-410-955-7810. as well as a study of the structure and dynamics of kDNA E-mail address: [email protected] (J.C. Morris). replication intermediates. We will ®rst review the properties

0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S0020-7519(01)00156-4 454 J.C. Morris et al. / International Journal for Parasitology 31 (2001) 453±458 2.1. Topoisomerase II

The ®rst mitochondrial replication enzyme puri®ed to homogeneity from C. fasciculata was a type II topoisomerase (topo II) (Melendy and Ray, 1989). This topo II is a homodimer of 132 kDa subunits. Like other enzymes of this type, it is ATP-dependent and catalyses catenation and decatenation of DNA in vitro. A homologue of the C. fasciculata enzyme has been cloned from Trypanosoma brucei (Strauss and Wang, 1990). This topoisomerase, as well as others characterised from T. brucei, is sensitive to many conventional topoisomerase inhibitors. These inhibitors, such as etoposide and VP16, have been valuable in studying enzyme function (Ray et al., 1992; Shapiro, 1994; Nenortas et al., 1998). A second topo II that has a distinct intramitochondrial localisation has been partially puri®ed from C. fasciculata (Shlomai et al., 1984). Fig. 1. EM showing a segment of a puri®ed C. fasciculata kinetoplast DNA network. Small loops are the 2.5 kb minicircles, and long strands threading 2.2. Universal minicircle sequence binding protein through the network interior are parts of the 38 kb maxicircles. EM by David PeÂrez-Morga. Part of the minicircle replication origin, the initiation site for leading strand synthesis, is a 12 nucleotide sequence of the characterised kDNA replication proteins and then known as the universal minicircle sequence (UMS). This describe our current model for kDNA replication. sequence is `universal' because it is found, with virtually no variation, in minicircles from all trypanosomatid species examined. A UMS binding protein (UMSBP) has been puri- 2. Proteins involved in kDNA replication and ®ed from C. fasciculata and is a homodimer of 13.7 kDa maintenance subunits (Tzfati et al., 1992). Amazingly, this protein also binds to DNA fragments containing a six nucleotide Replication proteins have been studied mainly in C. fasci- sequence (,80 nucleotides from the UMS) that serves as culata. This parasite is ideal for enzyme puri®cation and the initiation site for the ®rst Okazaki fragment (Abu-Elneel biochemical studies as it is non-pathogenic, it can be et al., 1999). This origin recognition protein, which likely grown in large quantities (up to 150 L, which yields ,400 plays a role in the initiation of minicircle replication, does g of cells) in inexpensive medium, and there is an ef®cient not bind to double-stranded oligonucleotides containing the method for isolating mitochondria (T. Saxowsky and M. UMS dodecamer or the hexameric sequence, although it Klingbeil, unpublished data). In this section we will discuss binds tightly and speci®cally to these sequences in single- the properties of the puri®ed enzymes and proteins. Later we stranded form (Abeliovich et al., 1993). Surprisingly, it does shall review their intramitochondrial localisation and spec- bind to these sequences in double-stranded form in cova- ulate on their function in kDNA replication. lently-closed intact free minicircles (Avrahami et al., 1995). Apparently, the minicircle sequence dictates some structural deformation in the origin region that allows binding (Avra- hami et al., 1995).

2.3. Primase

A 28 kDa protein that can synthesise small oligoribonu- cleotides (up to about 10 nucleotides in size) has been puri- ®ed from C. fasciculata mitochondria. The small RNAs that are products of this enzyme can prime Klenow DNA polymerase to initiate DNA synthesis in vitro (Li and Englund, 1997). Further characterisation of this enzyme is ongoing.

2.4. DNA polymerase b Fig. 2. Organisation of the kinetoplast DNA network in vivo. The C. fasciculata network is a disk 1 mm in diameter and 0.35 mm thick. The pie- A small (43 kDa) DNA polymerase b (pol b) has been shaped sector shows individual interlocked minicircles stretched out paral- puri®ed from C. fasciculata mitochondria (Torri and lel to the disk's axis. Englund, 1992). Biochemical studies indicate that the J.C. Morris et al. / International Journal for Parasitology 31 (2001) 453±458 455 enzyme is non-processive and, because it lacks a 30 proof- escence in situ hybridisation, there are also free minicircle reading exonuclease, error prone. However, pol b is ef®- replication intermediates in the antipodal sites (Ferguson et cient in ®lling small gaps (Torri et al., 1994). Sequence al., 1992). There is preliminary evidence that the second analysis indicates that this protein is related to mammalian topo II is localised throughout the kDNA disk (Shlomai, pol b (33% identical in sequence to the human enzyme). 1994). Primase is localised on the anterior and posterior This is the ®rst b-type polymerase described in mitochon- faces of the disk (Li and Englund, 1997). The protein loca- dria (Torri and Englund, 1995). Mammalian nuclear b poly- lisation diagrammed in Fig. 3 refers to cells undergoing merases and the yeast pol b homologue (Pol IV) function in kDNA replication. At other non-replicative stages of the base excision repair. The role of C. fasciculata mitochon- cell cycle some of the proteins alter their location (Johnson drial pol b is not fully understood, but the enzyme is prob- and Englund, 1998). ably not the major replicative polymerase (see below).

2.5. Ribonuclease H 4. kDNA replication intermediates

Structure-speci®c endonuclease 1 (SSE1) from C. fasci- The kDNA's network structure complicates its replica- culata mitochondria is a 32 kDa enzyme that has ribonu- tion mechanism. The problem is that each network, contain- clease H activity and may be involved in primer removal ing 5000 covalently-closed minicircles (in the case of C. (Engel and Ray, 1998). This protein has a domain similar to fasciculata), must double their minicircle copy number the 50 exonuclease domain of bacterial DNA polymerase I during each cell cycle. The two progeny networks must be (Engel and Ray, 1999). In vitro studies have revealed that distributed to the two daughter cells during cell division. SSE1 recognises the structure of its substrate, cleaving a There are serious topological problems that must be over- non-base-paired 50 tail on the 30 side of its ®rst base-paired come for this process to occur. In this review we will focus nucleotide (Engel and Ray, 1998). There are two other on the replication of minicircles, but see Hajduk et al. detectable RNase H activities (38 and 45 kDa) in C. fasci- (1984) and Carpenter and Englund (1995) for a description culata, of which the larger is enriched in puri®ed kineto- of maxicircle replication. plasts (Ray and Hines, 1995; Engel and Ray, 1998). Study of minicircle replication intermediates, mostly in Interestingly, a single gene (RNH1) encodes both of these C. fasciculata, has uncovered the following highlights of the proteins, which are not essential for cell viability as demon- replication mechanism. (A) Replication occurs during a strated by genetic knockout (Ray and Hines, 1995). These discrete phase of the cell cycle, nearly concurrent with the proteins can complement an Escherichia coli strain defec- nuclear S phase (Cosgrove and Skeen, 1970). (B) Prior to tive in RNase H, an enzyme implicated in the regulation of replication, minicircles are covalently closed, and after RNA priming (Campbell and Ray, 1993). replication they are gapped. The presence of gaps is thought to distinguish newly replicated minicircles from those that 2.6. p18, p17, and p16 have not been replicated, ensuring that each replicates once per cell cycle (Englund, 1978). (C) Minicircles do not repli- The C. fasciculata genes KAP2, KAP3, and KAP4 encode cate while linked to the network, but instead they are indi- the small basic proteins p18, p17, and p16, respectively (Xu vidually released from the network, presumably by a topo II and Ray, 1993; Xu et al., 1996). These proteins associate (Englund, 1979). (D) The covalently-closed free minicircles tightly with kDNA, as they were recovered from isolated replicate unidirectionally as u-structures, forming gapped kDNA networks after reversible cross-linking in vivo with progeny (Kitchin et al., 1984; Ntambi and Englund, 1985; formaldehyde (Xu and Ray, 1993). These histone-like proteins can condense kDNA in vitro and can rescue E. coli that are de®cient in the HU protein, a DNA-binding protein that plays a role in chromosomal condensation, replication, and recombination (Xu et al., 1996).

3. Localisation of kDNA replication proteins within the mitochondrial matrix

Immunoelectron and immuno¯uorescence microscopy have revealed speci®c localisation of these enzymes in distinct sites around the kinetoplast disk (see diagram in Fig. 3). The kDNA disk is ¯anked by two antipodal sites containing at least three enzymes involved in replication. These are topo II (Melendy et al., 1988), pol b (Ferguson et al., 1992), and SSE1 (Engel and Ray, 1998). Based on ¯uor- Fig. 3. Localisation of kinetoplast DNA replication enzymes. 456 J.C. Morris et al. / International Journal for Parasitology 31 (2001) 453±458 lently-closed minicircles shrinks. See Fig. 4 for a diagram of minicircle release and reattachment and Fig. 5 for evidence that newly replicated gapped minicircles are localised around the network periphery. (F) When all the minicircles have replicated, the minicircle copy number has doubled, to 10 000 in the case of C. fasciculata (PeÂrez-Morga and Englund, 1993b). At this time the gaps are repaired, the network undergoes scission, and the two networks, each containing a complete complement of covalently-closed minicircles, are distributed to the two daughter cells during cell division.

Fig. 4. Diagram of a replicating network and free minicircles, not drawn to 5. The current replication model scale. Covalently-closed minicircles are released from the network and undergo replication, forming two progeny containing gaps. These are reat- The diagram in Fig. 6 shows a section through the tached to the network periphery. The region of the network containing gapped minicircles is shown by dots. network with newly replicated and reattached minicircles (bold circles) indicated at the edges of the disk. The disk is ¯anked by the two antipodal sites and it is sandwiched by Birkenmeyer and Ray, 1986; Birkenmeyer et al., 1987). (E) the two zones of primase. Covalently-closed minicircles are Reattachment of the replicated gapped free minicircles released from the kDNA disk, possibly by the topo II that is occurs at the network periphery (Englund, 1978; Guilbride thought to reside in this region. Once released from the and Englund, 1998). This speci®city of free minicircle reat- network the free minicircles encounter primase and possibly tachment leads to the development of two zones in the other proteins such as UMSBP, helicases, and the replica- replicating network, a peripheral zone of newly replicated tive polymerase. There are two possibilities as to what could gapped minicircles and a central zone of covalently-closed happen next. The free minicircles could assemble into a minicircles. As replication proceeds the peripheral zone of replication initiation complex and migrate to the antipodal gapped minicircles enlarges and the central zone of cova- sites to complete replication. Alternatively, they could

Fig. 5. Isolated kinetoplast DNA networks visualised by ¯uorescence microscopy. (Left) Networks stained with 40,6-diamidino-2-phenylindole (DAPI). (Right) Same networks in which the gapped minicircles are labelled with ¯uorescein-deoxyuridine triphosphate (dUTP) using terminal transferase. Note the peripheral localisation of gapped minicircles. Networks with a narrow ring of ¯uorescein ¯uorescence are from early stages of replication. Those labelled uniformly with ¯uorescein have all minicircles replicated. Images by Lys Guilbride (Guilbride and Englund, 1998). J.C. Morris et al. / International Journal for Parasitology 31 (2001) 453±458 457 then be attached to the network periphery by a topo II and are completely repaired by pol b and a DNA ligase. There is a problem with this model. It predicts that progeny gapped free minicircles would be linked to the network only adjacent to the antipodal sites. Yet the ¯uorescence images shown in Fig. 5 clearly demonstrate that the gapped minicircle progeny are distributed uniformly around the network periphery. How does this uniform distribution occur? Several years ago we provided evidence that there is a relative movement of the kDNA disk and the antipodal sites that could easily account for the uniform distribution of the minicircles around the replicating network (PeÂrez- Morga and Englund, 1993a). One possibility, shown in Fig. 7, is that the kDNA disk actually spins, leading to a uniform distribution of minicircles around the disk.

6. What will happen in the new millennium? Fig. 6. Kinetoplast DNA replication model. See text for details. As discussed in this review, biochemical characterisation of C. fasciculata replication proteins in our lab and other complete replication before migrating to the antipodal sites labs has been a powerful method for elucidating the kDNA and the gapped progeny could then move to these sites. In replication mechanism. However, there could be problems both models, many of the minicircle gaps are repaired at the ahead if we continue to depend exclusively on this antipodal sites. This process could involve primer removal by SSE1 and gap ®lling by the pol b. The progeny mini- approach. For example, we found clear evidence that C. fasciculata mitochondria contain DNA polymerase activ- circles, still containing one or a small number of gaps, can ities in addition to the well-characterised pol b (M. Kling- beil, unpublished data), one of which could be the replicative polymerase. However, attempts to purify the polymerase were unsuccessful because of its instability. As an alternative, approaches based on genomics could be useful in the identi®cation of proteins involved in kDNA replication. Putative coding regions can be identi®ed in the rapidly advancing T. brucei genome project using homology-based searches. This sequence information is suf®cient for speci®c inhibition of gene expression utilising the recently developed technique of RNA interference (RNAi). This technique works well in T. brucei (Ngo et al., 1998; Wang et al., 2000). Genes responsible for pheno- types associated with kDNA can be cloned and recombinant proteins expressed in order to study their enzymatic properties and subcellular localisation. This coupling of genomic and proteomic strategies may provide the next advance in our understanding of the kDNA replication mechanism. Stay tuned.

Acknowledgements

Fig. 7. The spinning kinetoplast. The ellipse represents the top of the We thank Viiu Klein for valuable contributions to this kinetoplast disk. Small circles represent antipodal sites. Kinetoplast spins work. M.M.K. is supported by National Research Service in the direction of small arrows. Solid lines represent rows of minicircles Award Fellowship 5F32AI09789. T.T.S. is supported by the that are attached adjacent to antipodal sites. After nearly half a turn (lower right) the periphery is almost completely ®lled. Continued spinning of the Fannie and John Hertz Foundation. Work in our lab is kinetoplast results in minicircles being attached in a spiral pattern (PeÂrez- supported by grant GM 27608 from the National Institutes Morga and Englund, 1993a). of Health. 458 J.C. Morris et al. / International Journal for Parasitology 31 (2001) 453±458

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