In situ hybridization to the Crithidia fasciculata kinetoplast reveals two antipodal sites involved in kinetoplast DNA replication

Department of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland, United States
Cell (Impact Factor: 32.24). 09/1992; 70(4):621-9. DOI: 10.1016/0092-8674(92)90431-B
Source: PubMed


Kinetoplast DNA is a network of interlocked minicircles and maxicircles. In situ hybridization, using probes detected by digital fluorescence microscopy, has clarified the in vivo structure and replication mechanism of the network. The probe recognizes only nicked minicircles. Hybridization reveals prereplication kinetoplasts (with closed minicircles), donut-shaped replicating kinetoplasts (with nicked minicircles on the periphery and closed minicircles in the center), and postreplication kinetoplasts (with nicked minicircles). Replicating kinetoplasts are associated with two peripheral structures containing free minicircle replication intermediates and DNA polymerase. Replication may involve release of closed minicircles from the center of the kinetoplast and their migration to the peripheral structures, replication of the free minicircles therein, and then peripheral reattachment of the progeny minicircles to the kinetoplast.


Available from: Paul T Englund, Jan 06, 2015
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    • "brucei), used to kill tens of thousands of people each year and still puts about 70 million people at risk in many countries in the regions of sub-Saharan Africa [7] [12]. One important characteristic of Trypanosomes is the three dimensional organization of their mitochondrial DNA, known as kinetoplast DNA (kDNA), into several thousands of minicircles that are topologically linked forming a gigantic chainmail-like network that is confined in a cylindrically shaped region of the mitochondrion called kinetoplast disk ([6] [11]). Some topological properties of kDNA have been determined for Crithidia fasciculata , a model organism whose network is believed to mirror that of T. brucei. "
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    ABSTRACT: Trypanosomatida parasites, such as Trypanosoma and Leishmania, are the cause of deadly diseases in many third world countries. The three dimensional structure of their mitochondrial DNA, known as kinetoplast DNA (kDNA), is unique since it is organized into several thousands of minicircles that are topologically linked. How and why the minicircles form such a network have remained unanswered questions. In our previous work we have presented a model of network formation that hypothesizes that the network is solely driven by the confinement of minicircles. Our model shows that upon confinement a percolation network forms. This network grows into a space filling network, called saturation network, upon further confinement of minicircles. Our model also shows, in agreement with experimental data, that the mean valence of the network (that is, the average number of minicircles topologically linked to any minicircle in the network) grows linearly with minicircle density. In our previous studies however we disregarded DNA flexibility and used rigid minicircles to model DNA, here we address this limitation by allowing minicircles to be flexible. Our numerical results show that the topological characteristics that describe the growth and topology of the minicircle networks have similar values to those observed in the case of rigid minicircles suggesting that these properties are robust and therefore a potentially adequate description of the networks observed in Trypanosomatid parasites.
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    • "The recent report on the essential role of mitochondrial topoisomerase IA for minicircle theta structure resolution highlights the roles of kDNA modifying enzymes in network replication (Scocca and Shapiro, 2008). Okazaki fragment processing of progeny minicircles is mediated by structure-specific endonuclease 1, pol b, and DNA ligase kb at two antipodal sites flanking the kDNA disk (Ferguson et al., 1992; Engel and Ray, 1999; Hines et al., 2001; Downey et al., 2005). Minicircles that still contain at least one gap are subsequently reattached to the network periphery by TopoII localized at the antipodal sites (Melendy et al., 1988). "
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    ABSTRACT: The unique mitochondrial DNA of trypanosomes is a catenated network of minicircles and maxicircles called kinetoplast DNA (kDNA). The network is essential for survival, and requires an elaborate topoisomerase-mediated release and reattachment mechanism for minicircle theta structure replication. At least seven DNA polymerases (pols) are involved in kDNA transactions, including three essential proteins related to bacterial DNA pol I (POLIB, POLIC and POLID). How Trypanosoma brucei utilizes multiple DNA pols to complete the topologically complex task of kDNA replication is unknown. To fill this gap in knowledge we investigated the cellular role of POLIB using RNA interference (RNAi). POLIB silencing resulted in growth inhibition and progressive loss of kDNA networks. Additionally, unreplicated covalently closed precursors become the most abundant minicircle replication intermediate as minicircle copy number declines. Leading and lagging strand minicircle progeny similarly declined during POLIB silencing, indicating POLIB had no apparent strand preference. Interestingly, POLIB RNAi led to the accumulation of a novel population of free minicircles that is composed mainly of covalently closed minicircle dimers. Based on these data, we propose that POLIB performs an essential role at the core of the minicircle replication machinery.
    Molecular Microbiology 03/2010; 75(6):1414-25. DOI:10.1111/j.1365-2958.2010.07061.x · 4.42 Impact Factor
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    • "These proteins assemble a replication fork resulting in unidirectional θ-type replication. The two progeny minicircles are thought to migrate to the two antipodal sites, protein assemblies flanking the kDNA disk and positioned ∼180° apart (Ferguson et al., 1992). Within these sites are enzymes involved in later stages of minicircle replication including RNA primer removal and repair and ligation of most gaps between Okazaki fragments (Downey et al., 2005; Engel and Ray, 1998; Liu et al., 2005b; Torri and Englund, 1992). "
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    ABSTRACT: Kinetoplast DNA (kDNA), the trypanosome mitochondrial DNA, contains thousands of minicircles and dozens of maxicircles interlocked in a giant network. Remarkably, Trypanosoma brucei's genome encodes 8 PIF1-like helicases, 6 of which are mitochondrial. We now show that TbPIF2 is essential for maxicircle replication. Maxicircle abundance is controlled by TbPIF2 level, as RNAi of this helicase caused maxicircle loss, and its overexpression caused a 3- to 6-fold increase in maxicircle abundance. This regulation of maxicircle level is mediated by the TbHslVU protease. Previous experiments demonstrated that RNAi knockdown of TbHslVU dramatically increased abundance of minicircles and maxicircles, presumably because a positive regulator of their synthesis escaped proteolysis and allowed synthesis to continue. Here, we found that TbPIF2 level increases following RNAi of the protease. Therefore, this helicase is a TbHslVU substrate and an example of a positive regulator, thus providing a molecular mechanism for controlling maxicircle replication.
    Molecular cell 08/2009; 35(4):490-501. DOI:10.1016/j.molcel.2009.07.004 · 14.02 Impact Factor
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