Date of Award

2-2013

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Microbiology

First Advisor

Michele M. Klingbeil

Second Advisor

John M. Lopes

Third Advisor

Steven J. Sandler

Abstract

The mitochondrion contains its own genome. Replication of the mitochondrial DNA (mtDNA) is an essential process that, in most organisms, occurs through the cell cycle with no known mechanism to ensure spatial or temporal constrain. Failures to maintain mtDNA copy number affects cellular functions causing several human disorders. However, it is not clear how the cells control the mtDNA copy number. The mtDNA of trypanosomes, known as kinetoplast DNA (kDNA), is a structurally complex network of topologically interlocked DNA molecules (minicircles and maxicircles). The replication mechanism of the kDNA differs greatly with all other eukaryotic systems. Key features of the kDNA replication mechanism include defined regions for main replication events, coordination of a large number of proteins to drive the replication process, and replication once per cell cycle in near synchrony with nuclear S phase. Two main regions known as the kinetoflagellar zone (KFZ) and the antipodal sites are where main kDNA replication events are known to occur (i.e, initiation, DNA synthesis and Okazaki fragment processing). So far, the localization of the proteins involved in kDNA replication is restricted to two main regions: the KFZ and the antipodal sites. Three mechanisms that directly regulate kDNA replication proteins and serve to control kDNA replication have been proposed: (1) Reduction and oxidation status of the universal minicircle sequence binding protein (UMSBP) controls its binding to the origin sequence, (2) Trans-acting factors regulate the stability of mRNA encoding mitochondrial Topoisomerase II during the cell cycle and, (3) Regulation of TbPIF2 helicase protein levels by a HslVU-like protease to control maxicircle copy number. These mechanisms seem to be protein specific and it appears that a combination rather than a single mechanism regulates kDNA replication.

In this study we used Trypanosoma brucei to understand how mitochondrial DNA replication is controlled. We investigated the mechanism of how proteins transiently localize to the sites of DNA synthesis during cell cycle stages. Our data provides a comprehensive analysis of the first two examples of T. brucei kDNA replication proteins that have a cell cycle dependent localization (Ch. 2 and 3). The localization of two of the three essential mitochondrial DNA polymerases (TbPOLIC and TbPOLID) is under tight cell cycle control and not regulated by proteolysis. TbPOLIC and TbPOLID localize to the antipodal sites during kDNA S phase, however, at other cell cycle stages TbPOLIC becomes undetectable by immunofluorescent analysis and TbPOLID disperses through the mitochondrial matrix. In agreement with this data, TbPOLIC and TbPOLID replication complexes were not detected using affinity purification presumably because only a fraction of these proteins are participating in replication at a given time (Ch. 4). We propose that spatial and temporal changes in the dynamic localization of essential kDNA replication proteins provide a novel mechanism to control kDNA replication.

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