Mitochondria have their own closed circular genome. Mitochondrial DNA (mtDNA) has a 10 times greater rate of mutation than nuclear DNA. As DNA polymerase gamma is the only known DNA polymerase within the mitochondrion, it is essential for faithful replication, proofreading and repair of mtDNA. The overall goal of this proposal is to understand the fundamental mechanisms of exonuclease activity of DNA polymerase gamma in mtDNA metabolism, and to define its role in disease. Based on the following, we hypothesize that altered exonucleolytic activity of DNA polymerase gamma leads to mitochondrial dysfunction and disease. First, mutations in the exonuclease domain of DNA polymerase gamma cause devastating mitochondrial diseases. Second, polymerase domain mutations in DNA polymerase gamma that cause an error-prone phenotype can be ameliorated by functional exonuclease. Third, mouse models of proofreading-deficient DNA polymerase gamma show enhanced mtDNA mutations and accelerated ageing phenotype with reduced lifespan. Fourth, nucleoside reverse transcriptase inhibitors (NRTIs) used to control HIV infection induce mitochondrial dysfunction by inhibiting DNA polymerase gamma. Mitochondrial diseases can be caused by genetic and environmental factors. Unfortunately, there is no cure or no effective long-term treatment. As more diseases show mitochondrial defects, it is imperative that we understand how defects lead to disease. We will investigate the role of the exonuclease domain of DNA polymerase gamma in maintaining the integrity of mtDNA by:
AIM 1. To investigate the underiying consequences of disease mutations within the exonuclease domain, and how these give rise to mitochondrial dysfunction and disease.
AIM 2. To identify the critical regions within the exonuclease domain that are involved in proofreading, and to determine how efficientiy the exonuclease excises incorporated NRTIs, as well as nucleotides incorrectly incorporated opposite miscoding lesions in the template (such as 8oxoG, a marker of oxidative stress), and environmental DNA lesions (such as those caused by benzo[a]pyrene).

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Transition Award (R00)
Project #
5R00ES015555-03
Application #
7928234
Study Section
Special Emphasis Panel (NSS)
Program Officer
Reinlib, Leslie J
Project Start
2009-09-07
Project End
2012-07-31
Budget Start
2010-08-01
Budget End
2011-07-31
Support Year
3
Fiscal Year
2010
Total Cost
$249,000
Indirect Cost
Name
Medical University of South Carolina
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
183710748
City
Charleston
State
SC
Country
United States
Zip Code
29425
Chan, Sherine S L (2017) Inherited mitochondrial genomic instability and chemical exposures. Toxicology 391:75-83
Rahn, Jennifer J; Bestman, Jennifer E; Stackley, Krista D et al. (2015) Zebrafish lacking functional DNA polymerase gamma survive to juvenile stage, despite rapid and sustained mitochondrial DNA depletion, altered energetics and growth. Nucleic Acids Res 43:10338-52
Rahn, J J; Bestman, J E; Josey, B J et al. (2014) Novel Vitamin K analogs suppress seizures in zebrafish and mouse models of epilepsy. Neuroscience 259:142-54
Rahn, Jennifer J; Stackley, Krista D; Chan, Sherine S L (2013) Opa1 is required for proper mitochondrial metabolism in early development. PLoS One 8:e59218
Zhang, Linsheng; Chan, Sherine S L; Wolff, Daynna J (2011) Mitochondrial disorders of DNA polymerase ? dysfunction: from anatomic to molecular pathology diagnosis. Arch Pathol Lab Med 135:925-34
Stackley, Krista D; Beeson, Craig C; Rahn, Jennifer J et al. (2011) Bioenergetic profiling of zebrafish embryonic development. PLoS One 6:e25652
Chan, Sherine S L; Copeland, William C (2009) DNA polymerase gamma and mitochondrial disease: understanding the consequence of POLG mutations. Biochim Biophys Acta 1787:312-9
Kasiviswanathan, Rajesh; Longley, Matthew J; Chan, Sherine S L et al. (2009) Disease mutations in the human mitochondrial DNA polymerase thumb subdomain impart severe defects in mitochondrial DNA replication. J Biol Chem 284:19501-10
Chan, Sherine S L; Copeland, William C (2009) Functional analysis of mutant mitochondrial DNA polymerase proteins involved in human disease. Methods Mol Biol 554:59-72
Chan, Sherine S L; Naviaux, Robert K; Basinger, Alice A et al. (2009) De novo mutation in POLG leads to haplotype insufficiency and Alpers syndrome. Mitochondrion 9:340-5