Abstract
The mitochondrial genome represents a target for exogenous and endogenous damage. Its necessity for successful electron transport makes its repair valuable to the cell. Previous work from our lab has shown that mitochondrial DNA (mtDNA) can be repaired in mammalian cells, and the use of mitochondrial-targeted repair proteins can augment repair to enhance viability following genotoxic stress. In addition, it has also been shown that other repair enzymes that are targeted to the mitochondria can sensitize the cell to DNA damaging agents, thereby aiding the effectiveness of certain chemotherapeutic agents. The methods herein describe the development of mitochondrial-targeted proteins using plasmids or protein transduction domains. It includes the utilization of these constructs to create stably transfected cell lines, transiently transfected cell lines, viral-mediated transduction, and protein transduction domain-mediated mitochondrial protein localization. The end result will be a mammalian cell that expresses the mitochondrial-targeted protein of interest.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Driggers WJ, LeDoux SP, Wilson GL. (1993) Repair of oxidative damage within the mitochondrial DNA of RINr 38 cells. J Biol Chem 268, 22042–5.
Grishko V, Solomon M, Wilson GL, LeDoux SP, Gillespie MN. (2001) Oxygen radical-induced mitochondrial DNA damage and repair in pulmonary vascular endothelial cell phenotypes. Am J Physiol Lung Cell Mol Physiol 280, L1300–8.
Hollensworth SB, Shen C, Sim JE, Spitz DR, Wilson GL, LeDoux SP. (2000) Glial cell type-specific responses to menadione-induced oxidative stress. Free Radic Biol Med 28, 1161–74.
LeDoux SP, Patton NJ, Avery LJ, Wilson GL. (1993) Repair of N-methylpurines in the mitochondrial DNA of xeroderma pigmentosum complementation group D cells. Carcinogenesis 14, 913–7.
Ledoux SP, Shen CC, Grishko VI, Fields PA, Gard AL, Wilson GL. (1998) Glial cell-specific differences in response to alkylation damage. Glia 24, 304–12.
LeDoux SP, Wilson GL, Beecham EJ, Stevnsner T, Wassermann K, Bohr VA. (1992) Repair of mitochondrial DNA after various types of DNA damage in Chinese hamster ovary cells. Carcinogenesis 13, 1967–73.
Pettepher CC, LeDoux SP, Bohr VA, Wilson GL. (1991) Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the nitrosourea streptozotocin. J Biol Chem 266, 3113–7.
Shen CC, Wertelecki W, Driggers WJ, LeDoux SP, Wilson GL. (1995) Repair of mitochondrial DNA damage induced by bleomycin in human cells. Mutat Res 337, 19–23.
Wilson GL, Patton NJ, LeDoux SP. (1997) Mitochondrial DNA in beta-cells is a sensitive target for damage by nitric oxide. Diabetes 46, 1291–5.
Mandavilli BS, Santos JH, Van Houten B. (2002) Mitochondrial DNA repair and aging. Mutat Res 509,127–51.
Wilson DM, 3rd, Bohr VA. (2007) The mechanics of base excision repair, and its relationship to aging and disease. DNA Repair(Amst) 6, 544–59.
Dobson AW, Grishko V, LeDoux SP, Kelley MR, Wilson GL, Gillespie MN. (2002) Enhanced mtDNA repair capacity protects pulmonary artery endothelial cells from oxidant-mediated death. Am J Physiol Lung Cell Mol Physiol 283, L205–10.
Dobson AW, Xu Y, Kelley MR, LeDoux SP, Wilson GL. (2000) Enhanced mitochondrial DNA repair and cellular survival after oxidative stress by targeting the human 8-oxoguanine glycosylase repair enzyme to mitochondria. J Biol Chem 275, 37518–23.
Druzhyna NM, Hollensworth SB, Kelley MR, Wilson GL, Ledoux SP. (2003) Targeting human 8-oxoguanine glycosylase to mitochondria of oligodendrocytes protects against menadione-induced oxidative stress. Glia 42, 370–8.
Harrison JF, Hollensworth SB, Spitz DR, Copeland WC, Wilson GL, LeDoux SP. (2005) Oxidative stress-induced apoptosis in neurons correlates with mitochondrial DNA base excision repair pathway imbalance. Nucleic Acids Res 33, 4660–71.
Ho R, Rachek LI, Xu Y, Kelley MR, LeDoux SP, Wilson GL. (2007) Yeast apurinic/apyrimidinic endonuclease Apn1 protects mammalian neuronal cell line from oxidative stress. J Neurochem 102, 13–24.
Rachek LI, Grishko VI, Alexeyev MF, Pastukh VV, LeDoux SP, Wilson GL. (2004) Endonuclease III and endonuclease VIII conditionally targeted into mitochondria enhance mitochondrial DNA repair and cell survival following oxidative stress. Nucleic Acids Res 32, 3240–7.
Rachek LI, Grishko VI, Musiyenko SI, Kelley MR, LeDoux SP, Wilson GL. (2002) Conditional targeting of the DNA repair enzyme hOGG1 into mitochondria. J Biol Chem 277, 44932–7.
Rachek LI, Thornley NP, Grishko VI, LeDoux SP, Wilson GL. (2006) Protection of INS-1 cells from free fatty acid-induced apoptosis by targeting hOGG1 to mitochondria. Diabetes 55, 1022–8.
Shokolenko IN, Alexeyev MF, Robertson FM, LeDoux SP, Wilson GL. (2003) The expression of Exonuclease III from E. coli in mitochondria of breast cancer cells diminishes mitochondrial DNA repair capacity and cell survival after oxidative stress. DNA Repair(Amst) 2, 471–82.
Dobson AW, Kelley MR, Wilson GL, LeDoux SP. (2002) Targeting DNA repair proteins to mitochondria. Methods Mol Biol 197, 351–62.
Shokolenko IN, Alexeyev MF, LeDoux SP, Wilson GL. (2005) TAT-mediated protein transduction and targeted delivery of fusion proteins into mitochondria of breast cancer cells. DNA Repair(Amst) 4, 511–8.
Shimoda-Matsubayashi S, Matsumine H, Kobayashi T, Nakagawa-Hattori Y, Shimizu Y, Mizuno Y. (1996) Structural dimorphism in the mitochondrial targeting sequence in the human manganese superoxide dismutase gene. A predictive evidence for conformational change to influence mitochondrial transport and a study of allelic association in Parkinson's disease. Biochem Biophys Res Commun 226, 561–5.
Tamura T, McMicken HW, Smith CV, Hansen TN. (1996) Mitochondrial targeting of glutathione reductase requires a leader sequence. Biochem Biophys Res Commun 222, 659–63.
Vives E, Brodin P, Lebleu B. (1997) A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus. J Biol Chem 272, 16010–7.
Ho A, Schwarze SR, Mermelstein SJ, Waksman G, Dowdy SF. (2001) Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res 61, 474–7.
Wadia JS, Stan RV, Dowdy SF. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 10, 310–5.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Koczor, C.A., Snyder, J.W., Shokolenko, I.N., Dobson, A.W., Wilson, G.L., LeDoux, S.P. (2009). Targeting Repair Proteins to the Mitochondria of Mammalian Cells Through Stable Transfection, Transient Transfection, Viral Transduction, and TAT-Mediated Protein Transduction. In: Stuart, J.A. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 554. Humana Press. https://doi.org/10.1007/978-1-59745-521-3_15
Download citation
DOI: https://doi.org/10.1007/978-1-59745-521-3_15
Publisher Name: Humana Press
Print ISBN: 978-1-934115-60-2
Online ISBN: 978-1-59745-521-3
eBook Packages: Springer Protocols