|Michele M. Klingbeil
Assistant Professor of Microbiology, University of Massachusetts
Ph.D.: University of Toledo
Parasitology, the replication and repair of mitochondrial DNA (kinetoplast DNA network) in African trypanosomes
Trypanosomatid parasites (Trypanosoma and Leishmania species) cause deadly diseases in humans and livestock. Current treatments are often toxic and difficult to administer in endemic regions (mainly Third World countries). The need for new anti-trypanosomatid drugs has never been greater. As one of the earliest branching eukaryotic lineages,trypanosomes display a number of unusual biological properties. One property without counterpart in nature is their amazing mitochondrial DNA known as kinetoplast DNA (kDNA). The kDNA is a network containing thousands of catenated DNA molecules (minicircles and maxicircles, figure at left). We are interested in studying proteins involved in replicating this novel structure. Our primary focus is on a family of 4 DNA polymerases (Pol) from Trypanosoma brucei (Klingbeil et al., Mol. Cell 10, p. 175-186, figure at right) that are related to bacterial Pol I, all of which localize to the mitochondrion.This Pol I-like family is present in all the major trypanosomatid parasites, but not in mammals.
How is the kDNA network replicated?
Key to the mechanism of network replication is the release of minicircles from the kDNA network into the specialized kinetoflagellar zone to replicate as free circular molecules, and their subsequent reattachment at the network periphery. Several proteins localize to specific regions surrounding the condensed kDNA disk including 2 of the Pol I-like proteins (figure at right). Genetic studies using RNA interference (RNAi) indicate that Pol IB and Pol IC are involved in minicircle replication and essential for parasite survival. The other 2 polymerases could be involved in kDNA repair. In mammals, Pol gamma is the only DNA polymerase involved in mitochondrial DNA replication. Trypanosomatid parasites lack Pol gamma, but the Pol I-like family represents another intriguing property of trypanosomes different from the mammalian host, that serve as an attractive new drug target with broad applications to potentially treat several trypanosomatid parasitic diseases.
Using a combination of reverse genetics (RNAi), cell biology and biochemistry, we would like to answer several questions. What specific roles do the Pol I-like proteins play in replication and repair? What enzymatic properties distinguish between replication and repair functions? What other interacting proteins are required for kDNA replication and repair? Most importantly, are there inhibitors specific to these Pol I-like proteins?
El-Sayed, NMA, Myler PJ, Bartholomeu DC, Klingbeil, MM, Fraser, C, Stuart, KD and Andersson, B (2005) The genome sequence of Trypansoma cruzi, etiological agent of Chagas disease. Science 309 , 409-415. (Part of Special Science section)
Klingbeil, MM and Englund PT (2004) Closing the gaps in kinetoplast DNA replication. Proc. Natl. Acad. Sci. USA 10 , 4333-4334.
Saxowsky, TM, Choudhary, G, Klingbeil, MM and Englund PT (2003) “Trypanosoma brucei has two distinct mitochondrial DNA polymerase beta enzymes.” J. Biol. Chem. 278 (49), 49095-49101.
Klingbeil, MM, Motyka,SA, and Englund PT (2002) “Multiple mitochondrial DNA polymerases from Trypanosoma brucei.” Mol. Cell 10 (1), 175-186 (July, Cover image).
Klingbeil, MM, Drew, ME, Liu, Y, Morris, JC, Motyka, SA, Saxowsky, TT, Wang, Z, and Englund PT (2001) “Unlocking the secrets of trypanosome kinetoplast DNA network replication.” Protist 152 (4), 255-262.
Morris, JC, Drew, ME, Klingbeil, MM, Motyka, SA, Saxowsky, TT, Wang, Z, and Englund, PT (2001) Replication of kinetoplast DNA: an update for the new millennium. Intl. J. Parasitol. 31 , 453-458.