The mitochondrial DNA is 16 times more prone to oxidative damage and evolves 10-20 times faster than nuclear DNA. Point mutations and deletions in this mitochondrial genome give rise to a wide range of diseases, such as Leber's hereditary optic neuropathy, retinitis pigmentosa, Kearn-Sayre syndrome, Pearson marrow pancreas syndrome and ocular myopathy. These mutations may occur during replication by the DNA polymerase gamma. The DNA polymerase gamma differs form the nuclear DNA polymerases due to its sensitivity to antiviral nucleotide analogs, such as AZT and dideoxynucleotides. Patients treated with AZT develop a ragged-red fiber myopathy associated with a reduction in mitochondrial DNA levels. How the mitochondrial DNA polymerase makes point and deletion mutations and what structural properties set this polymerase apart from the nuclear DNA polymerases to give rise to its inhibition patterns are poorly understood. To address these questions we have cloned the DNA polymerase gamma genes and cDNA from S. pombe, D. melanogaster and Homo Sapiens. The recombinant human mitochondrial DNA polymerase gamma protein has been functionally overexpressed greater than 100 fold in insect cells by a recombinant baculovirus and in E. coli. The overexpressed protein is currently being purified to homogeneity and enzymatically characterized. Two mutant DNA polymerase gamma proteins were made by site-specific mutageneisis; an exonuclease deficient and a dideoxy-resistent DNA polymerase. Antibodies against the human pol gamma were used to study the regulation of polymerase gamma expression in human cells in the presence and absence of mitochondrial DNA in human cells. DNA polymerase gamma protein and mRNA levels in mitochondrial deficient cells were the same as their parental cells, suggesting no feedback control from the mitochondria to the nucleus.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES065078-03
Application #
2574428
Study Section
Special Emphasis Panel (LMG)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
1996
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Sharma, Nidhi; Chakravarthy, Srinivas; Longley, Matthew J et al. (2018) The C-terminal tail of the NEIL1 DNA glycosylase interacts with the human mitochondrial single-stranded DNA binding protein. DNA Repair (Amst) 65:11-19
Krasich, Rachel; Copeland, William C (2017) DNA polymerases in the mitochondria: A critical review of the evidence. Front Biosci (Landmark Ed) 22:692-709
DeBalsi, Karen L; Hoff, Kirsten E; Copeland, William C (2017) Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases. Ageing Res Rev 33:89-104
Prasad, Rajendra; Ça?layan, Melike; Dai, Da-Peng et al. (2017) DNA polymerase ?: A missing link of the base excision repair machinery in mammalian mitochondria. DNA Repair (Amst) 60:77-88
DeBalsi, Karen L; Longley, Matthew J; Hoff, Kirsten E et al. (2017) Synergistic Effects of the in cis T251I and P587L Mitochondrial DNA Polymerase ? Disease Mutations. J Biol Chem 292:4198-4209
Varma, Hemant; Faust, Phyllis L; Iglesias, Alejandro D et al. (2016) Whole exome sequencing identifies a homozygous POLG2 missense variant in an infant with fulminant hepatic failure and mitochondrial DNA depletion. Eur J Med Genet 59:540-5
Copeland, William C; Kasiviswanathan, Rajesh; Longley, Matthew J (2016) Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase ?. Methods Mol Biol 1351:19-26
Young, Matthew J; Copeland, William C (2016) Human mitochondrial DNA replication machinery and disease. Curr Opin Genet Dev 38:52-62
Copeland, William C; Longley, Matthew J (2014) Mitochondrial genome maintenance in health and disease. DNA Repair (Amst) 19:190-8
Copeland, William C (2014) Defects of mitochondrial DNA replication. J Child Neurol 29:1216-24

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