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Replication of Kinetoplast DNA: an Update for the New Millennium

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Replication of Kinetoplast DNA: an Update for the New Millennium 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 intramito- chondrial 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 topoisome- rase (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. fasci- culata 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 intramito- chondrial 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 poly- merase 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. fasci- culata 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.
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