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Robert L. Low |
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. In the past 2 years, my lab has finally succeeded in developing a faithful in vitro assay system to study the mechanism of H-strand priming.
Our recent results with this asssay indicate that the primer for H-strand replication is synthesized by a novel Ori H directed DNA primase and not by the mtDNA RNA polymerase as widely assumed. In addition to our work with this primase, a potent endonuclease identified in crude fractions of mt protein has been purified and shown to attack an unusual sequence tract near Ori H. We are currently exploring the possibility that the activity is the nicking component of a novel site-specific swivelase required for DNA replication and/or an activity which eliminates oxidatively damaged genomes.
Recent Publications:
