Abstract
The mitochondrial single-stranded DNA-binding protein (mtSSB) regulates the function of the mitochondrial DNA (mtDNA) replisome. In vitro, mtSSB stimulates the activity of enzymatic components of the replisome, namely mtDNA helicase and DNA polymerase gamma (Pol γ). We have demonstrated that the stimulatory properties of mtSSB result from its ability to organize the single-stranded DNA template in a specific manner. Here we present methods employing electron microscopy and enzymatic assays to characterize and classify the mtSSB-DNA complexes and their effects on the activity of Pol γ.
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References
Ciesielski GL, Oliveira MT, Kaguni LS (2016) Animal mitochondrial DNA replication. Enzyme 39:255–292
Korhonen JA, Pham XH, Pellegrini M, Falkenberg M (2004) Reconstitution of a minimal mtDNA replisome in vitro. EMBO J 23:2423–2429
Cerrón F, de Lorenzo S, Lemishko KM, Ciesielski GL, Kaguni LS, Cao FJ, Ibarra B (2019) Replicative DNA polymerases promote active displacement of SSB proteins during lagging strand synthesis. Nucleic Acids Res 47:5723–5734
Morin JA, Cerrón F, Jarillo J, Beltran-Heredia E, Ciesielski GL, Arias-Gonzalez JR, Kaguni LS, Cao FJ, Ibarra B (2017) DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein. Nucleic Acids Res 45:7237–7248
Piro-Mégy C, Sarzi E, Tarrés-Solé A, Péquignot M, Hensen F, Quilès M, Manes G, Chakraborty A, Sénéchal A, Bocquet B, Cazevieille C, Roubertie A, Müller A, Charif M, Goudenège D, Lenaers G, Wilhelm H, Kellner U, Weisschuh N, Wissinger B, Zanlonghi X, Hamel C, Spelbrink JN, Solà M, Delettre C (2020) Dominant mutations in mtDNA maintenance gene SSBP1 cause optic atrophy and foveopathy. J Clin Invest 130(1):143–156
Gustafson MA, McCormick EM, Perera L, Longley MJ, Bai R, Kong J, Dulik M, Shen L, Goldstein AC, McCormack SE, Laskin BL, Leroy BP, Ortiz-Gonzalez XR, Ellington MG, Copeland WC, Falk MJ (2019) Mitochondrial single-stranded DNA binding protein novel de novo SSBP1 mutation in a child with single large-scale mtDNA deletion (SLSMD) clinically manifesting as Pearson, Kearns-Sayre, and Leigh syndromes. PLoS One 14:e0221829
Del Dotto V, Ullah F, Di Meo I, Magini P, Gusic M, Maresca A, Caporali L, Palombo F, Tagliavini F, Baugh EH, Macao B, Szilagyi Z, Péron C, Gustafson MA, Khan K, La Morgia C, Barboni P, Carbonelli M, Valentino ML, Liguori R, Shashi V, Sullivan JA, Nagaraj S, El-Dairi M, Iannaccone A, Cutcutache I, Bertini E, Carrozzo R, Emma F, Diomedi-Camassei F, Zanna C, Armstrong M, Page MJ, Boesch S, Wortmann SB, Kopajtich R, Stong N, Sperl W, Davis E, Copeland WC, Seri M, Falkenberg M, Prokisch H, Katsanis N, Tiranti V, Pippucci T, Carelli V (2020) SSBP1 mutations cause mtDNA depletion underlying a complex optic atrophy disorder. J Clin Invest 130(1):108–125
Korhonen JA, Gaspari M, Falkenberg M (2003) TWINKLE Has 5′ -> 3′ DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein. J Biol Chem 278:48627–48632
Oliveira MT, Kaguni LS (2010) Functional roles of the N- and C-terminal regions of the human mitochondrial single-stranded DNA-binding protein. PLoS One 5:e15379
Oliveira MT, Kaguni LS (2011) Reduced stimulation of recombinant DNA polymerase γ and mitochondrial DNA (mtDNA) helicase by variants of mitochondrial single-stranded DNA-binding protein (mtSSB) correlates with defects in mtDNA replication in animal cells. J Biol Chem 286:40649–40658
Ciesielski GL, Bermek O, Rosado-Ruiz FA, Hovde SL, Neitzke OJ, Griffith JD, Kaguni LS (2015) Mitochondrial single-stranded DNA-binding proteins stimulate the activity of DNA polymerase γ by organization of the template DNA. J Biol Chem 290:28697–28707
Clayton DA (1982) Replication of animal mitochondrial DNA. Cell 28:693–705
Miralles Fusté J, Shi Y, Wanrooij S, Zhu X, Jemt E, Persson Ö, Sabouri N, Gustafsson CM, Falkenberg M (2014) In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of DNA replication. PLoS Genet 10:e1004832
Farr CL, Matsushima Y, Lagina AT, Luo N, Kaguni LS (2004) Physiological and biochemical defects in functional interactions of mitochondrial DNA polymerase and DNA-binding mutants of single-stranded DNA-binding protein. J Biol Chem 279:17047–17053
Griffith JD, Christiansen G (1978) Electron microscope visualization of chromatin and other DNA-protein complexes. Annu Rev Biophys Bioeng 7:19–35
Acknowledgments
G.L.C. and O.B. would like to acknowledge Dr. Laurie S. Kaguni and Dr. Jack D. Griffith, who mentored the authors in the described methodology during their postdoctoral training. G.L.C. was partially supported by a grant from the Auburn University at Montgomery Research Grant-in-Aid Program.
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Bermek, O., Ciesielski, G.L. (2021). Analysis of Mitochondrial SSB-DNA Complexes and Their Effects on DNA Polymerase γ Activity by Electron Microscopy and Enzymatic Assays. In: Oliveira, M.T. (eds) Single Stranded DNA Binding Proteins. Methods in Molecular Biology, vol 2281. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1290-3_16
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DOI: https://doi.org/10.1007/978-1-0716-1290-3_16
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