Mitochondrial DNA Polymerase Structure and Function

State University New York Stony Brook, Stony Brook NY

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): Human mitochondrial DNA (mtDNA) is a 16,569 base pair genome that encodes 13 protein subunits of respiratory complexes and a set of rRNA and tRNA genes required for their translation in the organelle. Point mutations and deletions in mtDNA cause a number of human diseases, many of which present as neuromuscular disorders. Mutations in mtDNA are also thought to contribute to common disorders such as Parkinson's disease and Huntington's disease and possibly to the aging process. All genes involved in maintenance of mtDNA are encoded in the nucleus and their protein products are imported into mitochondria. Mutations in mtDNA reflect errors in the replication and repair of the genome introduced by DNA polymerase gamma (gamma), which has two subunits, a 137-kDa catalytic subunit, pol gammaA, and an accessory subunit, pol gammaB. DNA pol gamma is also a target for toxic side effects of nucleoside inhibitors used in therapy for HIV. Our laboratory has recently cloned the accessory factors for Xenopus, mouse and human pol gamma and provided the first evidence that this acts as a processivity factor. We have recently determined the crystal structure of mouse pot gammaB, which shows a remarkable similarity to bacterial tRNA synthetases. The proposed research will build upon our experience with the structure of the small subunit to study the structure of the catalytic subunit and of the entire holoenzyme. A detailed understanding of the structure of the pot gamma holoenzyme and its contacts with accessory replication factors is essential to appreciate its central rote in generating large deletions. We propose to compare the structure of the human enzyme to that of pot gamma in yeasts where the catalytic subunit evidently does not employ a processivity factor. We will search for proteins that interact tightly with pol gamma subunits. Other experiments will introduce site-directed mutations into human pol gammaB based upon the known structure to study its ability to dimerize, to act as a processivity factor, and to bind nucleic acids. We will test the hypothesis that the nucleic acid binding properties of pol gammaB help to direct the polymerase to sites of replication initiation.