MOLECULAR CHARACTERIZATION OF THE MITOCHONDRIAL DNA POLYMERASE

Environmental Health Sciences

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Abstract

Summary of Work: More than 1 in 4,000 children born in the United States each year will develop a mitochondrial disease by age 10 with a mortality rate from 10 to 50 percent. Defects in mitochondrial function have been linked to several of the most common diseases of aging. Over 50 million people in the US suffer from chronic degenerative disorders involving mitochondria of compromised function. The mutation rate of the mitochondrial genome is 10-20 times greater than in the nuclear DNA. Mutations within mitochondrial DNA are the main cause of these mitochondrial diseases. These mutations can arise during replication of the mitochondrial DNA. The goal of this project is to understand the contribution by the replication apparatus to the production and prevention of mutations in mitochondrial DNA. Our enzymatic and structural studies of the mitochondrial DNA replication proteins are addressing these goals. We previously cloned the human DNA polymerase gamma and accessory subunit. The human catalytic and accessory subunits have been overproduced in baculovirus and E. coli, respectively. The fidelity of the DNA polymerase gamma complex was studied in detail. The majority of errors made by Pol g are ?1 frameshifts. Pol g makes a high number of +1 base addition errors and is the first example of a pol I type polymerase that does dislocation mutagenesis. The p55 accessory subunit lowers the fidelity at a single base pair by five-fold and increased frameshifts at least three-fold in homopolymeric runs. We have developed a new assay to identify new nuclear genes that affect mitochondrial mutation rates in S. cerevisiae. Disruption of one of these genes, Mcg1 (Mitochondrial copper gene 1), results in a 51-fold higher mutation rate, slow growth, increased sensitivity to copper, increased accumulation of copper in the mitochondria, and increased formation of petite colonies. These results indicate that Mcg1 can play an important role in mitochondrial copper regulation and free radical detoxification. A human homolog was identified.