Direct monitoring of the stepwise condensation of kinetoplast DNA networks.

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.

Interactions of a replication initiator with histone H1-like proteins remodel the condensed mitochondrial genome.

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, consists of several thousand topologically interlocked DNA circles. Mitochondrial histone H1-like proteins were implicated in the condensation of kDNA into a nucleoid structure in the mitochondrial matrix. However, the mechanism that remodels kDNA, promoting its accessibility to the replication machinery, has not yet been described. Analyses, using yeast two hybrid system, co-immunoprecipitation, and protein-protein cross-linking, revealed specific protein-protein interactions between the kDNA replication initiator protein universal minicircle sequence-binding protein (UMSBP) and two mitochondrial histone H1-like proteins. Fluorescence and electron microscopy, as well as biochemical analyses, demonstrated that these protein-protein interactions result in the decondensation of kDNA. UMSBP-mediated decondensation rendered the kDNA network accessible to topological decatenation by topoisomerase II, yielding free kDNA minicircle monomers. Hence, UMSBP has the potential capacity to function in vivo in the activation of the prereplication release of minicircles from the network, a key step in kDNA replication, which precedes and enables its replication initiation. These observations demonstrate the prereplication remodeling of a condensed mitochondrial DNA, which is mediated via specific interactions of histone-like proteins with a replication initiator, rather than through their posttranslational covalent modifications.

Trypanosomes have six mitochondrial DNA helicases with one controlling kinetoplast maxicircle replication.

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.

Enzymatic mechanism controls redox-mediated protein-DNA interactions at the replication origin of kinetoplast DNA minicircles.

Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomatids. Its major components are several thousand topologically interlocked DNA minicircles. Their replication origins are recognized by universal minicircle sequence-binding protein (UMSBP), a CCHC-type zinc finger protein, which has been implicated with minicircle replication initiation and kDNA segregation. Interactions of UMSBP with origin sequences in vitro have been found to be affected by the protein's redox state. Reduction of UMSBP activates its binding to the origin, whereas UMSBP oxidation impairs this activity. The role of redox in the regulation of UMSBP in vivo was studied here in synchronized cell cultures, monitoring both UMSBP origin binding activity and its redox state, throughout the trypanosomatid cell cycle. These studies indicated that UMSBP activity is regulated in vivo through the cell cycle dependent control of the protein's redox state. The hypothesis that UMSBP's redox state is controlled by an enzymatic mechanism, which mediates its direct reduction and oxidation, was challenged in a multienzyme reaction, reconstituted with pure enzymes of the trypanosomal major redox-regulating pathway. Coupling in vitro of this reaction with a UMSBP origin-binding reaction revealed the regulation of UMSBP activity through the opposing effects of tryparedoxin and tryparedoxin peroxidase. In the course of this reaction, tryparedoxin peroxidase directly oxidizes UMSBP, revealing a novel regulatory mechanism for the activation of an origin-binding protein, based on enzyme-mediated reversible modulation of the protein's redox state. This mode of regulation may represent a regulatory mechanism, functioning as an enzyme-mediated, redox-based biological switch.