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
Protozoan parasites in the family Trypanosomatidae are
early diverging eukaryotes that cause important tropical
diseases including African sleeping sickness, leishmaniasis,
and Chagas' disease in humans as well as nagana in African
livestock. All of the trypanosomatid parasites have a
remarkable mitochondrial DNA, termed kinetoplast DNA
(kDNA), that has a structure unlike that of any other
known DNA in nature. Within the matrix of each cell's
single mitochondrion the kDNA is a network of a few thousand topologically interlocked DNA circles. There are two
types of circles, maxicircles and minicircles. Each network
contains several dozen maxicircles (in most species they
range in size from about 20 to 40 kb) and several thousand
minicircles (usually 0.5±2.5 kb, although in some species
they are larger). For a more comprehensive review on
kDNA see Shapiro and Englund (1995). Like mitochondrial
DNAs from mammalian cells or yeast, maxicircles encode
ribosomal RNAs and some of the proteins required for mitochondrial bioenergetic processes. Some RNA transcripts of
maxicircles are post-transcriptionally modi®ed by the insertion or deletion of uridine residues to form functional open
reading frames, a process termed RNA editing. Editing
speci®city is directed by guide RNAs that are encoded by
the minicircles. For a review on editing see Estevez and
Simpson (1999).
* Corresponding author. Tel.: 11-410-955-3458; fax: 11-410-955-7810.
E-mail address: [email protected] (J.C. Morris).
Most studies of kDNA replication in our laboratory, the
Ray laboratory (UCLA) and the Shlomai laboratory
(Hebrew University) have focused on the insect parasite
Crithidia fasciculata. Crithidia fasciculata kDNA networks
puri®ed from non-replicating cells are remarkably homogeneous in size and shape, being planar, elliptically-shaped
structures about 10 by 15 mm in size (see EM in Fig. 1
showing a segment of an isolated kDNA network). All of
the minicircles are covalently closed, relaxed, and linked to
an average of three neighbouring minicircles by single interlocks (Rauch et al., 1993; Chen et al., 1995). Topologically,
the network has a striking resemblance to the chain mail of
medieval armour. Within the parasite's single mitochondrion, the network is condensed in a highly ordered fashion
into a disk-shaped structure about 1 mm in diameter and
0.35 mm thick. (Fig. 2 illustrates how the kDNA is
condensed into a disk.) The kDNA disk is always positioned
near the basal body of the ¯agellum and perpendicular to the
axis of the ¯agellum. Remarkably, there is evidence for a
direct physical linkage between the basal body and the
kDNA network, even though these two structures are separated by the double membrane of the mitochondrion (Robinson and Gull, 1991).
In this review we describe the replication of kDNA, a
subject that our lab has studied for many years. Our knowledge of kDNA 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 kDNA
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: S 0020-751 9(01)00156-4
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J.C. Morris et al. / International Journal for Parasitology 31 (2001) 453±458
2.1. Topoisomerase II
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
through the network interior are parts of the 38 kb maxicircles. EM by
David PeÂrez-Morga.
of the characterised kDNA replication proteins and then
describe our current model for kDNA replication.
2. Proteins involved in kDNA replication and
maintenance
Replication proteins have been studied mainly in C. fasciculata. This parasite is ideal for enzyme puri®cation and
biochemical studies as it is non-pathogenic, it can be
grown in large quantities (up to 150 L, which yields ,400
g of cells) in inexpensive medium, and there is an ef®cient
method for isolating mitochondria (T. Saxowsky and M.
Klingbeil, unpublished data). In this section we will discuss
the properties of the puri®ed enzymes and proteins. Later we
shall review their intramitochondrial localisation and speculate on their function in kDNA replication.
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).
2.2. Universal minicircle sequence binding protein
Part of the minicircle replication origin, the initiation site
for leading strand synthesis, is a 12 nucleotide sequence
known as the universal minicircle sequence (UMS). This
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®ed from C. fasciculata and is a homodimer of 13.7 kDa
subunits (Tzfati et al., 1992). Amazingly, this protein also
binds to DNA fragments containing a six nucleotide
sequence (,80 nucleotides from the UMS) that serves as
the initiation site for the ®rst Okazaki fragment (Abu-Elneel
et al., 1999). This origin recognition protein, which likely
plays a role in the initiation of minicircle replication, does
not bind to double-stranded oligonucleotides containing the
UMS dodecamer or the hexameric sequence, although it
binds tightly and speci®cally to these sequences in singlestranded form (Abeliovich et al., 1993). Surprisingly, it does
bind to these sequences in double-stranded form in covalently-closed intact free minicircles (Avrahami et al., 1995).
Apparently, the minicircle sequence dictates some structural
deformation in the origin region that allows binding (Avrahami et al., 1995).
2.3. Primase
A 28 kDa protein that can synthesise small oligoribonucleotides (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 pieshaped sector shows individual interlocked minicircles stretched out parallel to the disk's axis.
A small (43 kDa) DNA polymerase b (pol b) has been
puri®ed from C. fasciculata mitochondria (Torri and
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 3 0 proofreading exonuclease, error prone. However, pol b is ef®cient in ®lling small gaps (Torri et al., 1994). Sequence
analysis indicates that this protein is related to mammalian
pol b (33% identical in sequence to the human enzyme).
This is the ®rst b-type polymerase described in mitochondria (Torri and Englund, 1995). Mammalian nuclear b polymerases and the yeast pol b homologue (Pol IV) function in
base excision repair. The role of C. fasciculata mitochondrial pol b is not fully understood, but the enzyme is probably not the major replicative polymerase (see below).
escence in situ hybridisation, there are also free minicircle
replication intermediates in the antipodal sites (Ferguson et
al., 1992). There is preliminary evidence that the second
topo II is localised throughout the kDNA disk (Shlomai,
1994). Primase is localised on the anterior and posterior
faces of the disk (Li and Englund, 1997). The protein localisation diagrammed in Fig. 3 refers to cells undergoing
kDNA replication. At other non-replicative stages of the
cell cycle some of the proteins alter their location (Johnson
and Englund, 1998).
2.5. Ribonuclease H
4. kDNA replication intermediates
Structure-speci®c endonuclease 1 (SSE1) from C. fasciculata mitochondria is a 32 kDa enzyme that has ribonuclease H activity and may be involved in primer removal
(Engel and Ray, 1998). This protein has a domain similar to
the 5 0 exonuclease domain of bacterial DNA polymerase I
(Engel and Ray, 1999). In vitro studies have revealed that
SSE1 recognises the structure of its substrate, cleaving a
non-base-paired 5 0 tail on the 3 0 side of its ®rst base-paired
nucleotide (Engel and Ray, 1998). There are two other
detectable RNase H activities (38 and 45 kDa) in C. fasciculata, of which the larger is enriched in puri®ed kinetoplasts (Ray and Hines, 1995; Engel and Ray, 1998).
Interestingly, a single gene (RNH1) encodes both of these
proteins, which are not essential for cell viability as demonstrated by genetic knockout (Ray and Hines, 1995). These
proteins can complement an Escherichia coli strain defective in RNase H, an enzyme implicated in the regulation of
RNA priming (Campbell and Ray, 1993).
The kDNA's network structure complicates its replication mechanism. The problem is that each network, containing 5000 covalently-closed minicircles (in the case of C.
fasciculata), must double their minicircle copy number
during each cell cycle. The two progeny networks must be
distributed to the two daughter cells during cell division.
There are serious topological problems that must be overcome for this process to occur. In this review we will focus
on the replication of minicircles, but see Hajduk et al.
(1984) and Carpenter and Englund (1995) for a description
of maxicircle replication.
Study of minicircle replication intermediates, mostly in
C. fasciculata, has uncovered the following highlights of the
replication mechanism. (A) Replication occurs during a
discrete phase of the cell cycle, nearly concurrent with the
nuclear S phase (Cosgrove and Skeen, 1970). (B) Prior to
replication, minicircles are covalently closed, and after
replication they are gapped. The presence of gaps is thought
to distinguish newly replicated minicircles from those that
have not been replicated, ensuring that each replicates once
per cell cycle (Englund, 1978). (C) Minicircles do not replicate while linked to the network, but instead they are individually released from the network, presumably by a topo II
(Englund, 1979). (D) The covalently-closed free minicircles
replicate unidirectionally as u-structures, forming gapped
progeny (Kitchin et al., 1984; Ntambi and Englund, 1985;
2.6. p18, p17, and p16
The C. fasciculata genes KAP2, KAP3, and KAP4 encode
the small basic proteins p18, p17, and p16, respectively (Xu
and Ray, 1993; Xu et al., 1996). These proteins associate
tightly with kDNA, as they were recovered from isolated
kDNA networks after reversible cross-linking in vivo with
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.
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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
scale. Covalently-closed minicircles are released from the network and
undergo replication, forming two progeny containing gaps. These are reattached to the network periphery. The region of the network containing
gapped minicircles is shown by dots.
Birkenmeyer and Ray, 1986; Birkenmeyer et al., 1987). (E)
Reattachment of the replicated gapped free minicircles
occurs at the network periphery (Englund, 1978; Guilbride
and Englund, 1998). This speci®city of free minicircle reattachment leads to the development of two zones in the
replicating network, a peripheral zone of newly replicated
gapped minicircles and a central zone of covalently-closed
minicircles. As replication proceeds the peripheral zone of
gapped minicircles enlarges and the central zone of cova-
5. The current replication model
The diagram in Fig. 6 shows a section through the
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
the two zones of primase. Covalently-closed minicircles are
released from the kDNA disk, possibly by the topo II that is
thought to reside in this region. Once released from the
network the free minicircles encounter primase and possibly
other proteins such as UMSBP, helicases, and the replicative polymerase. There are two possibilities as to what could
happen next. The free minicircles could assemble into a
replication initiation complex and migrate to the antipodal
sites to complete replication. Alternatively, they could
Fig. 5. Isolated kinetoplast DNA networks visualised by ¯uorescence microscopy. (Left) Networks stained with 4 0 ,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 (PeÂrezMorga 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.
Fig. 6. Kinetoplast DNA replication model. See text for details.
complete replication before migrating to the antipodal sites
and the gapped progeny could then move to these sites. In
both models, many of the minicircle gaps are repaired at the
antipodal sites. This process could involve primer removal
by SSE1 and gap ®lling by the pol b. The progeny minicircles, still containing one or a small number of gaps, can
6. What will happen in the new millennium?
As discussed in this review, biochemical characterisation
of C. fasciculata replication proteins in our lab and other
labs has been a powerful method for elucidating the kDNA
replication mechanism. However, there could be problems
ahead if we continue to depend exclusively on this
approach. For example, we found clear evidence that C.
fasciculata mitochondria contain DNA polymerase activities in addition to the well-characterised pol b (M. Klingbeil, 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 phenotypes 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
kinetoplast disk. Small circles represent antipodal sites. Kinetoplast spins
in the direction of small arrows. Solid lines represent rows of minicircles
that are attached adjacent to antipodal sites. After nearly half a turn (lower
right) the periphery is almost completely ®lled. Continued spinning of the
kinetoplast results in minicircles being attached in a spiral pattern (PeÂrezMorga and Englund, 1993a).
We thank Viiu Klein for valuable contributions to this
work. M.M.K. is supported by National Research Service
Award Fellowship 5F32AI09789. T.T.S. is supported by the
Fannie and John Hertz Foundation. Work in our lab is
supported by grant GM 27608 from the National Institutes
of Health.
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J.C. Morris et al. / International Journal for Parasitology 31 (2001) 453±458
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