Recent data indicate that accumulate damage to mitochondrial DNA (mtDNA) may play an important role in cardiac myopathy, stroke , and other age-related pathologies such as neurodegeneration. In fact, it has been proposed that the accumulation of mtDNA damage plays a fundamental role in normal aging. In addition, a human genetic disease (autosomal dominant progressive external ophthalmoplegia, adPEO) has recently been described in which mtDNA instability can lead to premature death. These observations highlight the deleterious health consequences of damage to mtDNA. Yet comparatively little is known about DNA repair of mtDNA in mammalian cells. A series of recent observations have provided new information about this process: (1) A novel mitochondrial-specific DNA ligase has been identified, (2) Mitochondrial protein extracts prepared from a variety of mammalian cells possess potent DNA end-joining activity, (3) The products of this end-joining activity bear a striking resemblance to mutant mitochondrial DNA molecules observed in vivo, and (4) A DNA end- binding activity has been identified in mitochondrial protein extracts. Based on these observations, we have hypothesized that mammalian mitochondrial possess a non-homologous end-joining DNA repair pathway, analogous to that which functions in the nucleus. The experiments described in this proposal are designed to test this hypothesis. Specifically, gene targeting will be used to inactivate the mitochondrial DNA ligase gene, and the mtDNA repair/stability phenotype of 'knockout' cells evaluated. In addition, the mitochondrial DNA end-binding gene will be cloned. Gene targeting will be used to create mutant cell lines in which this gene has been inactivated, and the mtDNA repair and stability phenotype of these cells will be determined. Based on preliminary results presented in this proposal, it is reasonable to predict that the knockout cell lines described above could possess a mtDNA mutator phenotype. If this provides to be the case, future studies could be devoted to the creation of similar gene inactivations in mice, thereby permitting a test of the hypothesis that accumulated mtDNA damage influences aging.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG016678-04
Application #
6629826
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Mccormick, Anna M
Project Start
2000-04-15
Project End
2005-03-31
Budget Start
2003-04-15
Budget End
2005-03-31
Support Year
4
Fiscal Year
2003
Total Cost
$191,803
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Pharmacology
Type
Schools of Medicine
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Donahue, Sarah L; Tabah, Azah A; Schmitz, Kyle et al. (2007) Defective signal joint recombination in fanconi anemia fibroblasts reveals a role for Rad50 in V(D)J recombination. J Mol Biol 370:449-58
De, Ananya; Campbell, Colin (2007) A novel interaction between DNA ligase III and DNA polymerase gamma plays an essential role in mitochondrial DNA stability. Biochem J 402:175-86
De, Ananya; Donahue, Sarah L; Tabah, Azah et al. (2006) A novel interaction [corrected] of nucleolin with Rad51. Biochem Biophys Res Commun 344:206-13
Donahue, Sarah L; Lundberg, Richard; Campbell, Colin (2004) Intermediate DNA repair activity associated with the 322delG allele of the fanconi anemia complementation group C gene. J Mol Biol 342:1443-55
Donahue, Sarah L; Lundberg, Richard; Saplis, Rachel et al. (2003) Deficient regulation of DNA double-strand break repair in Fanconi anemia fibroblasts. J Biol Chem 278:29487-95
Bordone, Laura; Campbell, Colin (2002) DNA ligase III is degraded by calpain during cell death induced by DNA-damaging agents. J Biol Chem 277:26673-80
Donahue, Sarah L; Campbell, Colin (2002) A DNA double strand break repair defect in Fanconi anemia fibroblasts. J Biol Chem 277:46243-7
Usachev, Yuriy M; DeMarco, Steven J; Campbell, Colin et al. (2002) Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4. Neuron 33:113-22
Lundberg, R; Mavinakere, M; Campbell, C (2001) Deficient DNA end joining activity in extracts from fanconi anemia fibroblasts. J Biol Chem 276:9543-9
Donahue, S L; Corner, B E; Bordone, L et al. (2001) Mitochondrial DNA ligase function in Saccharomyces cerevisiae. Nucleic Acids Res 29:1582-9

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