@article {1414, title = {Mitochondrial heat shock protein machinery hsp70/hsp40 is indispensable for proper mitochondrial DNA maintenance and replication.}, journal = {MBio}, volume = {6}, year = {2015}, month = {2015}, abstract = {

UNLABELLED: Mitochondrial chaperones have multiple functions that are essential for proper functioning of mitochondria. In the human-pathogenic protist Trypanosoma brucei, we demonstrate a novel function of the highly conserved machinery composed of mitochondrial heat shock proteins 70 and 40 (mtHsp70/mtHsp40) and the ATP exchange factor Mge1. The mitochondrial DNA of T. brucei, also known as kinetoplast DNA (kDNA), is represented by a single catenated network composed of thousands of minicircles and dozens of maxicircles packed into an electron-dense kDNA disk. The chaperones mtHsp70 and mtHsp40 and their cofactor Mge1 are uniformly distributed throughout the single mitochondrial network and are all essential for the parasite. Following RNA interference (RNAi)-mediated depletion of each of these proteins, the kDNA network shrinks and eventually disappears. Ultrastructural analysis of cells depleted for mtHsp70 or mtHsp40 revealed that the otherwise compact kDNA network becomes severely compromised, a consequence of decreased maxicircle and minicircle copy numbers. Moreover, we show that the replication of minicircles is impaired, although the lack of these proteins has a bigger impact on the less abundant maxicircles. We provide additional evidence that these chaperones are indispensable for the maintenance and replication of kDNA, in addition to their already known functions in Fe-S cluster synthesis and protein import.

IMPORTANCE: Impairment or loss of mitochondrial DNA is associated with mitochondrial dysfunction and a wide range of neural, muscular, and other diseases. We present the first evidence showing that the entire mtHsp70/mtHsp40 machinery plays an important role in mitochondrial DNA replication and maintenance, a function likely retained from prokaryotes. These abundant, ubiquitous, and multifunctional chaperones share phenotypes with enzymes engaged in the initial stages of replication of the mitochondrial DNA in T. brucei.

}, issn = {2150-7511}, doi = {10.1128/mBio.02425-14}, author = {T{\'y}{\v c}, Ji{\v r}{\'\i} and Klingbeil, Michele M and Luke{\v s}, Julius} } @article {382, title = {Mitochondrial DNA polymerase POLIB is essential for minicircle DNA replication in African trypanosomes.}, journal = {Mol Microbiol}, volume = {75}, year = {2010}, month = {2010 Mar}, pages = {1414-25}, 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.}, keywords = {DNA Polymerase beta, DNA Replication, DNA, Kinetoplast, Gene Silencing, Protozoan Proteins, Trypanosoma brucei brucei}, issn = {1365-2958}, doi = {10.1111/j.1365-2958.2010.07061.x}, author = {Bruhn, David F and Mozeleski, Brian and Falkin, Laurie and Klingbeil, Michele M} } @article {388, title = {Multiple mitochondrial DNA polymerases in Trypanosoma brucei.}, journal = {Mol Cell}, volume = {10}, year = {2002}, month = {2002 Jul}, pages = {175-86}, abstract = {Kinetoplast DNA (kDNA), the unusual mitochondrial DNA of Trypanosoma brucei, is a network containing thousands of catenated circles. Database searching for a kDNA replicative polymerase (pol) revealed no mitochondrial pol gamma homolog. Instead, we identified four proteins (TbPOLIA, IB, IC, and ID) related to bacterial pol I. Remarkably, all four localized to the mitochondrion. TbPOLIB and TbPOLIC localized beside the kDNA where replication occurs, and their knockdown by RNA interference caused kDNA network shrinkage. Furthermore, silencing of TbPOLIC caused loss of both minicircles and maxicircles and accumulation of minicircle replication intermediates, consistent with a role in replication. While typical mitochondria contain one DNA polymerase, pol gamma, trypanosome mitochondria contain five such enzymes, including the previously characterized pol beta.}, keywords = {Amino Acid Sequence, Animals, DNA, Kinetoplast, DNA-Directed DNA Polymerase, Mitochondria, Molecular Sequence Data, Phylogeny, Protein Structure, Tertiary, Protein Transport, RNA, Double-Stranded, RNA, Messenger, Sequence Homology, Amino Acid, Trypanosoma brucei brucei}, issn = {1097-2765}, author = {Klingbeil, Michele M and Motyka, Shawn A and Englund, Paul T} }