Klingbeil DNA Replication Lab

Research


	Trypanosomatid Mitochondrial DNA Replication Trypanosoma brucei survival requires the kDNA network, therefore understanding the replication and repair mechanisms of this unusual DNA structure is an important aspect of trypanosome biology. For this single celled eukaryote, kDNA network replication requires the coordinated replication of each maxicircle and minicircle in close synchrony with nuclear S phase. The topological constraints of replicating an interlocked DNA network dictates some unusual features such as the elaborate topoisomerase II-mediated release-and-reattachment mechanism for minicircle replication, and at least seven mitochondrial DNA polymerases (pols) for kDNA transactions (see figure below right).
	The main project of the lab examines a family of four mitochondrial DNA polymerases related to bacterial DNA pol I that appear to have specialized functions in kDNA replication and repair. Multiple DNA pols in mitochondrial DNA replication have never been documented in any other eukaryote. We have already determined that three of the pol I-like proteins are essential for parasite viability making them drug target candidates. This NIH funded project is focused on three main areas: a) clarification of the in vivo roles using genetic tools such as RNAi, b) characterization of the enzymatic properties of the individual DNA pols, and c) identification of interacting proteins.
	Nuclear DNA Replication Initiation While studies on the replication of the mitochondrial kDNA network are more advanced, very little information is available about trypanosomatid nuclear DNA replication mechanisms or the machinery involved in replication initiation. Surprisingly, in contrast to the six-subunit (Orc1-6) Origin Recognition Complex (ORC) found in all other model eukaryotes, trypanosomatid genomes predict just a single ORC subunit, Orc1. Our second research area investigates the function of this core DNA replication initiation protein using RNAi, and provides information on the understudied area of DNA replication initiation machinery in trypanosomatids. Another goal of this research area is to identify other protein components that might participate in origin binding.