Abstract
Condensation and remodeling of nuclear genomes play an essential role in the regulation of gene expression and replication. Yet, our understanding of these processes and their regulatory role in other DNA-containing organelles, has been limited. This study focuses on the packaging of kinetoplast DNA (kDNA), the mitochondrial genome of kinetoplastids. Severe tropical diseases, affecting large human populations and livestock, are caused by pathogenic species of this group of protists. kDNA consists of several thousand DNA minicircles and several dozen DNA maxicircles that are linked topologically into a remarkable DNA network, which is condensed into a mitochondrial nucleoid. In vitro analyses implicated the replication protein UMSBP in the decondensation of kDNA, which enables the initiation of kDNA replication. Here, we monitored the condensation of kDNA, using fluorescence and atomic force microscopy. Analysis of condensation intermediates revealed that kDNA condensation proceeds via sequential hierarchical steps, where multiple interconnected local condensation foci are generated and further assemble into higher order condensation centers, leading to complete condensation of the network. This process is also affected by the maxicircles component of kDNA. The structure of condensing kDNA intermediates sheds light on the structural organization of the condensed kDNA network within the mitochondrial nucleoid.
Highlights
Tropical diseases, such as African sleeping sickness, South and Central American Chagas disease and the Leishmaniases, affecting large human populations and livestock, are caused by infection of parasitic protists of the group Trypanosomatidae
As we have previously shown, while each one of the four KAPs of C. fasciculata has the capacity to condense kinetoplast DNA (kDNA), only KAP3 and KAP4 proteins interact with universal minicircle sequence binding protein (UMSBP), and these specific protein–protein interactions lead to the remodeling of KAP3/KAP4-condensed kDNA n etworks[21]
We have reported that an order of magnitude higher concentration of UMSBP was required, in order to decondense kDNA networks that were condensed by the human histone H1 protein, compared to the UMSBP concentrations needed for decondensation of kDNA networks, condensed by KAP321
Summary
Tropical diseases, such as African sleeping sickness, South and Central American Chagas disease and the Leishmaniases, affecting large human populations and livestock, are caused by infection of parasitic protists of the group Trypanosomatidae. Condensation of genomes limits their accessibility to the replication and transcription machineries This has been studied extensively in the case of nuclear chromatin, revealing that uncoiling of the nucleosomal complex, known as chromatin remodeling, is mediated mainly by histone posttranslational modifications, which decrease their affinity to D NA22. Electron microscopy (EM) analyses revealed that isolated kDNA networks form an elliptical cup-shaped structure, with a major axis of 15 μm and a minor axis of 10 μm[9] As suggested by these analyses, the kDNA network condenses in vivo into a disc structure of 1.0 × 0.4 μm[10,11], in which the interlocked minicircles are stretched out and stand side by side. These studies have suggested that UMSBP-KAP3/4 interactions may play an important role in the pre-replication decondensation of kDNA, by enabling the release of minicircles from the network, and thereby the activation of kDNA replication initiation[21]
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