: Kinetoplast DNA (kDNA) is the mitochondrial DNA of trypanosomes, parasitic protozoa that cause major human disease. The major subject of our study is Trypanosoma brucei that causes African sleeping sickness; kDNA is an unusual structure, consisting of DNA circles that are catenated into a giant network. The network contains several thousand minicircles and a few dozen maxicircles. The subject of this proposal is the replication of kDNA. Major steps in kDNA synthesis include the sequential release of minicircles from the network by a topoisomerase to form free minicircles, replication of the free minicircles via theta structures, reattachment of the progeny minicircles to the network, and division of the double-size network into two daughter networks. The major emphasis of this proposal is to discover new proteins involved in kDNA replication. Because of the unusual structure of kDNA and because it is essential for parasite viability, these proteins could be targets for anti-trypanosome chemotherapy. One approach for discovering new kDNA replication proteins, in Specific Aim 1, is to screen an RNA interference library for cells with depleted kDNA, and preliminary data show that this approach is feasible. A second approach, in Specific Aim 2, is based on proteomics, in which mitochondrial DNA binding proteins are identified by tandem mass spectrometry. Again, preliminary data indicate that this strategy is also feasible. The proposed experiments in Specific Aim 3 are to characterize and determine the function of proteins discovered in Aims 1 and 2. Characterization will involve study of enzymatic properties and intracellular localization. Functional analysis will depend heavily on gene silencing by RNAi and an analysis of changes in structure of kDNA networks and free minicircle replication intermediates. ? ?

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
Project #
Application #
Study Section
Tropical Medicine and Parasitology Study Section (TMP)
Program Officer
Mcgugan, Glen C
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Johns Hopkins University
Schools of Medicine
United States
Zip Code
Kammers, Kai; Cole, Robert N; Tiengwe, Calvin et al. (2015) Detecting Significant Changes in Protein Abundance. EuPA Open Proteom 7:11-19
Wang, Jianyang; Pappas-Brown, Valeria; Englund, Paul T et al. (2014) TbKAP6, a mitochondrial HMG box-containing protein in Trypanosoma brucei, is the first trypanosomatid kinetoplast-associated protein essential for kinetoplast DNA replication and maintenance. Eukaryot Cell 13:919-32
Povelones, Megan L; Tiengwe, Calvin; Gluenz, Eva et al. (2013) Mitochondrial shape and function in trypanosomes requires the outer membrane protein, TbLOK1. Mol Microbiol 87:713-29
Jensen, Robert E; Englund, Paul T (2012) Network news: the replication of kinetoplast DNA. Annu Rev Microbiol 66:473-91
Wang, Jianyang; Englund, Paul T; Jensen, Robert E (2012) TbPIF8, a Trypanosoma brucei protein related to the yeast Pif1 helicase, is essential for cell viability and mitochondrial genome maintenance. Mol Microbiol 83:471-85
Clayton, April M; Guler, Jennifer L; Povelones, Megan L et al. (2011) Depletion of mitochondrial acyl carrier protein in bloodstream-form Trypanosoma brucei causes a kinetoplast segregation defect. Eukaryot Cell 10:286-92
Gluenz, Eva; Povelones, Megan L; Englund, Paul T et al. (2011) The kinetoplast duplication cycle in Trypanosoma brucei is orchestrated by cytoskeleton-mediated cell morphogenesis. Mol Cell Biol 31:1012-21
Roy Chowdhury, Arnab; Bakshi, Rahul; Wang, Jianyang et al. (2010) The killing of African trypanosomes by ethidium bromide. PLoS Pathog 6:e1001226
Liu, Beiyu; Yildirir, Gokben; Wang, Jianyang et al. (2010) TbPIF1, a Trypanosoma brucei mitochondrial DNA helicase, is essential for kinetoplast minicircle replication. J Biol Chem 285:7056-66
Liu, Beiyu; Wang, Jianyang; Yaffe, Nurit et al. (2009) Trypanosomes have six mitochondrial DNA helicases with one controlling kinetoplast maxicircle replication. Mol Cell 35:490-501

Showing the most recent 10 out of 23 publications