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This laboratory studies the mechanism of mitochondrial (mt) DNA replication in the mammalian cell. MtDNA is a 16 kb, circular duplex DNA which encodes 13 polypeptide subunits of the oxidative phosphorylation pathway of the inner membrane of the mitochondrion. This mini chromosome is maintained in all cells through repeated cycles of DNA synthesis to a level of a few to several thousand copies. Our goal is to understand how a new cycle of mtDNA synthesis is first initiated from the origin of mtDNA replication and to eventually learn how the process of mtDNA synthesis is controlled and regulated in response to the growth and metabolic needs of the cell. In the past few years, we have begun to realize how important the replication of mtDNA is to the health and long term survival of the cell. Replication produces the multiple copies of the genome which every cell needs in order to assemble a functional respiratory chain for the synthesis of ATP. As well, replication replaces the mtDNA circles that are continually being destroyed from the high level of oxidative damage incurred from the respiratory chain. Although mtDNA replication usually proceeds normally, under some circumstances it produces errors, creating mutations in key genes. The accumulation of such defects in mtDNA may be important and contribute to aging, neurodegenerative disease and possibly even neoplasia.

For several years it has been known that replication of the mt genome begins with the synthesis of one of the two strands of the DNA duplex, the so-called heavy (H)-strand, from a specific site on the genome known as the origin of H-strand replication (Ori H). Since the mtDNA polymerase can not initiate new DNA chains de novo, synthesis of this H-strand is dependent upon the production of an Ori H-specific oligonucleotide primer. Although the creation of this primer represents the first and one of the most important steps in mtDNA replication, the enzymes and DNA binding proteins that orchestrate its synthesis have until now remained elusive. Recently, my lab has finally succeeded in developing a faithful in vitro assay system to study the mechanism of H-strand priming.

Our ongoing results with this assay system suggest that the primer for H-strand replication can be synthesized by a novel origin directed DNA primase and not exclusively by the mtDNA RNA polymerase as widely presumed. In addition, we are currently studying a newly discovered mitochondrial DNA topoisomerase II activity, and other mitochondrial enzyme activities that could help repair oxidative damage incurred in mtDNA in vivo.