Errors in mitochondrial DNA replication have been hypothesized to drive aging and age-associated disease. The best evidence to date of a causal role for mitochondrial mutation in aging is the finding of an accelerated aging phenotype in mice expressing an error-prone form of the mitochondrial DNA polymerase; however, it is challenging to interpret data from studies performed with proteins having compromised function. Accordingly, I propose to develop a mitochondrial DNA polymerase with enhanced replication fidelity. An anti-mutator mitochondrial polymerase will serve as a model to determine the true role of mitochondrial DNA mutation in health and lifespan.
In Specific Aim 1, 1 will create specific mutations in the mouse mitochondrial DNA polymerase, utilizing known anti-mutator substitutions that have been characterized in prokaryotic model systems, and determine the effect of these substitutions on polymerase fidelity.
In Specific Aim 2, 1 will analyze the most promising anti-mutator polymerase mutants by quantitatively determining their activity, error rate, mutation spectrum, and biochemical properties.
In Specific Aim 3, 1 will determine the in vivo consequences of enhanced fidelity of mitochondrial DNA synthesis by expressing the anti-mutator polymerases in eukaryotic cells. A better understanding of the role of mitochondrial DNA replication fidelity in health and lifespan will lend insight into the fundamental mechanisms underlying aging itself. Because a large number of human diseases increase in both incidence and severity as a function of age, a better understanding of the aging process may result in strategies to delay or prevent the occurrence of disease. ? ? ?
| Schmitt, Michael W; Venkatesan, Ranga N; Pillaire, Marie-Jeanne et al. (2010) Active site mutations in mammalian DNA polymerase delta alter accuracy and replication fork progression. J Biol Chem 285:32264-72 |
| Bielas, Jason H; Schmitt, Michael W; Icreverzi, Amalia et al. (2009) Molecularly evolved thymidylate synthase inhibits 5-fluorodeoxyuridine toxicity in human hematopoietic cells. Hum Gene Ther 20:1703-7 |
| Schmitt, Michael W; Matsumoto, Yoshihiro; Loeb, Lawrence A (2009) High fidelity and lesion bypass capability of human DNA polymerase delta. Biochimie 91:1163-72 |
