The mitochondrion represents a target of reactive oxygen stress and mitochondrial DNA appears to be an early and sensitive marker of this stress. Many human diseases are associated with reactive oxygen, including cancer, heart disease and neurodegenerative diseases. Mitochondria are essential organelles for generating ATP during oxidative phosphorylation. The mitochondrial DNA encodes 13 polypeptides, eleven are involved in electron transport and two serve as subunits of ATP synthase. Damage to mitochondrial DNA is repaired, but prolonged oxidant treatment results in persistent mtDNA damage, loss of mitochondrial function, increase in p21Waf1/CIP, and apoptosis. These observations suggest that mitochondrial injury, specifically DNA damage, is important for reactive oxygen- induced toxicity. We are testing the hypothesis that reactive oxygen species (ROS) generated in the mitochondria result in mtDNA damage, which in turn causes the release of more ROS (superoxide, hydrogen peroxide, and the hydroxyl radical) that lead to further mitochondrial decline and many degenerative diseases associated with aging. In collaboration with Dr. Steve Kleeberger, hyperoxia is being used to look at the induction of mitochondrial DNA damage. ? ? During the past year we have been investigating the nature of double-strand break repair in highly purified mitochondrial extracts from human cells. We have developed a highly sensitive, quantitativePCR-based DSB repair assay. Utilizing this assay, we observe the repair of restriction endonuclease-induced DSBs catalyzed by highly purified mitochondrial extracts. DNA containing cohesive ends (5 or 3 overhangs) is repaired more efficiently than blunt-ended DNA (6.6%, 4.1% and 1.5% repaired, respectively). To elucidate the mechanism of mitochondrial DSB repair, we further investigated the rejoining of PstI-generated DSBs. This DSB repair is coupled with the processing of DNA ends, resulting in the loss of approximately 50 bases surrounding the PstI site. Sequence analysis revealed several patterns of the repaired DNA, most with deletions spanning 4-7 bp direct repeats. We hypothesize that mitochondrial nucleases resect the DNA to reveal short stretches of homology thus allowing annealing and ligation of broken DNA. The nucleases responsible for DNA resection are being investigated. This type of mtDNA repair would lead to the loss of expression of critical mitochondrial encoded proteins.? ? There is an ever increasing number of neurodegenerative diseases and mitochondrial myopathies associated with alterations in the mitochondrial genome. One clinical manifestation associated with the loss of mtDNA between direct repeats is Kearns-Sayre Syndrome. Our study of mitochondrial DSB repair might shed new light on the underlying mechanism of this and other mitochondrial associated diseases.? ? ? Finally we are also looking at another system in which we have in collaboration with Christi Walter, have engineered a mitochondrially-targeted EcoRI construct that is under the control of doxycycline, such that withdraw of DOX leads to its expression in both tissue culture cells and in mice.

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National Institute of Environmental Health Sciences (NIEHS)
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Ma, Wenjian; Panduri, Vijayalakshmi; Sterling, Joan F et al. (2009) The transition of closely opposed lesions to double-strand breaks during long-patch base excision repair is prevented by the coordinated action of DNA polymerase delta and Rad27/Fen1. Mol Cell Biol 29:1212-21
Wielgus, Albert R; Chignell, Colin F; Miller, David S et al. (2007) Phototoxicity in human retinal pigment epithelial cells promoted by hypericin, a component of St. John's wort. Photochem Photobiol 83:706-13
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Santos, Janine Hertzog; Meyer, Joel N; Van Houten, Bennett (2006) Mitochondrial localization of telomerase as a determinant for hydrogen peroxide-induced mitochondrial DNA damage and apoptosis. Hum Mol Genet 15:1757-68
Santos, Janine H; Meyer, Joel N; Mandavilli, Bhaskar S et al. (2006) Quantitative PCR-based measurement of nuclear and mitochondrial DNA damage and repair in mammalian cells. Methods Mol Biol 314:183-99
Stuart, Gregory R; Santos, Janine H; Strand, Micheline K et al. (2006) Mitochondrial and nuclear DNA defects in Saccharomyces cerevisiae with mutations in DNA polymerase gamma associated with progressive external ophthalmoplegia. Hum Mol Genet 15:363-74
Van Houten, Bennett; Woshner, Victoria; Santos, Janine H (2006) Role of mitochondrial DNA in toxic responses to oxidative stress. DNA Repair (Amst) 5:145-52
Fariss, Marc W; Chan, Catherine B; Patel, Manisha et al. (2005) Role of mitochondria in toxic oxidative stress. Mol Interv 5:94-111
Mandavilli, Bhaskar S; Boldogh, Istvan; Van Houten, Bennett (2005) 3-nitropropionic acid-induced hydrogen peroxide, mitochondrial DNA damage, and cell death are attenuated by Bcl-2 overexpression in PC12 cells. Brain Res Mol Brain Res 133:215-23
Santos, Janine Hertzog; Meyer, Joel N; Skorvaga, Milan et al. (2004) Mitochondrial hTERT exacerbates free-radical-mediated mtDNA damage. Aging Cell 3:399-411

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