Abstract
Mitochondrial DNA is replicated by the nuclear-encoded DNA polymerase γ (pol γ) which is composed of a single 140 kDa catalytic subunit and a dimeric 55 kDa accessory subunit. Mitochondrial DNA is vulnerable to various forms of damage, including several types of oxidative lesions, UV-induced photoproducts, chemical adducts from environmental sources, as well as alkylation and inter-strand cross-links from chemotherapy agents. Although many of these lesions block DNA replication, pol γ can bypass some lesions by nucleotide incorporation opposite a template lesion and further extension of the DNA primer past the lesion. This process of translesion synthesis (TLS) by pol γ can occur in either an error-free or an error-prone manner. Assessment of TLS requires extensive analysis of oligonucleotide substrates and replication products by denaturing polyacrylamide sequencing gels. This chapter presents protocols for the analysis of translesion DNA synthesis.
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References
Kukat C, Wurm CA, Spahr H, Falkenberg M, Larsson NG, Jakobs S (2011) Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA. Proc Natl Acad Sci U S A 108:13534–13539
Cline SD (2012) Mitochondrial DNA damage and its consequences for mitochondrial gene expression. Biochim Biophys Acta 1819:979–991
Copeland WC, Longley MJ (2014) Mitochondrial genome maintenance in health and disease. DNA Repair (Amst) 19:190–198
Kanuri M, Minko IG, Nechev LV, Harris TM, Harris CM, Lloyd RS (2002) Error prone translesion synthesis past gamma-hydroxypropano deoxyguanosine, the primary acrolein-derived adduct in mammalian cells. J Biol Chem 277:18257–18265
Minko IG, Washington MT, Kanuri M, Prakash L, Prakash S, Lloyd RS (2003) Translesion synthesis past acrolein-derived DNA adduct, gamma-hydroxypropanodeoxyguanosine, by yeast and human DNA polymerase eta. J Biol Chem 278:784–790
Washington MT, Minko IG, Johnson RE, Wolfle WT, Harris TM, Lloyd RS, Prakash S, Prakash L (2004) Efficient and error-free replication past a minor-groove DNA adduct by the sequential action of human DNA polymerases iota and kappa. Mol Cell Biol 24:5687–5693
Washington MT, Minko IG, Johnson RE, Haracska L, Harris TM, Lloyd RS, Prakash S, Prakash L (2004) Efficient and error-free replication past a minor-groove N2-guanine adduct by the sequential action of yeast Rev1 and DNA polymerase zeta. Mol Cell Biol 24:6900–6906
Wolfle WT, Johnson RE, Minko IG, Lloyd RS, Prakash S, Prakash L (2005) Human DNA polymerase iota promotes replication through a ring-closed minor-groove adduct that adopts a syn conformation in DNA. Mol Cell Biol 25:8748–8754
McCulloch SD, Kokoska RJ, Garg P, Burgers PM, Kunkel TA (2009) The efficiency and fidelity of 8-oxo-guanine bypass by DNA polymerases delta and eta. Nucleic Acids Res 37:2830–2840
McCulloch SD, Kokoska RJ, Masutani C, Iwai S, Hanaoka F, Kunkel TA (2004) Preferential cis-syn thymine dimer bypass by DNA polymerase eta occurs with biased fidelity. Nature 428:97–100
Takata K, Arana ME, Seki M, Kunkel TA, Wood RD (2010) Evolutionary conservation of residues in vertebrate DNA polymerase N conferring low fidelity and bypass activity. Nucleic Acids Res 38:3233–3244
Stone JE, Kumar D, Binz SK, Inase A, Iwai S, Chabes A, Burgers PM, Kunkel TA (2011) Lesion bypass by S. cerevisiae Pol zeta alone. DNA Repair (Amst) 10:826–834
Graziewicz MA, Longley MJ, Copeland WC (2006) DNA polymerase gamma in mitochondrial DNA replication and repair. Chem Rev 106:383–405
Lee YS, Kennedy WD, Yin YW (2009) Structural insight into processive human mitochondrial DNA synthesis and disease-related polymerase mutations. Cell 139:312–324
Lee YS, Lee S, Demeler B, Molineux IJ, Johnson KA, Yin YW (2010) Each monomer of the dimeric accessory protein for human mitochondrial DNA polymerase has a distinct role in conferring processivity. J Biol Chem 285:1490–1499
Graziewicz MA, Bienstock RJ, Copeland WC (2007) The DNA polymerase gamma Y955C disease variant associated with PEO and parkinsonism mediates the incorporation and translesion synthesis opposite 7,8-dihydro-8-oxo-2′-deoxyguanosine. Hum Mol Genet 16:2729–2739
Graziewicz MA, Sayer JM, Jerina DM, Copeland WC (2004) Nucleotide incorporation by human DNA polymerase gamma opposite benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyguanosine and deoxyadenosine. Nucleic Acids Res 32:397–405
Kasiviswanathan R, Gustafson MA, Copeland WC, Meyer JN (2012) Human mitochondrial DNA polymerase gamma exhibits potential for bypass and mutagenesis at UV-induced cyclobutane thymine dimers. J Biol Chem 287:9222–9229
Kasiviswanathan R, Minko IG, Lloyd RS, Copeland WC (2013) Translesion synthesis past acrolein-derived DNA adducts by human mitochondrial DNA polymerase gamma. J Biol Chem 288:14247–14255
Kasiviswanathan R, Longley MJ, Young MJ, Copeland WC (2010) Purification and functional characterization of human mitochondrial DNA polymerase gamma harboring disease mutations. Methods 51:379–384
Lim SE, Ponamarev MV, Longley MJ, Copeland WC (2003) Structural determinants in human DNA polymerase gamma account for mitochondrial toxicity from nucleoside analogs. J Mol Biol 329:45–57
Longley MJ, Ropp PA, Lim SE, Copeland WC (1998) Characterization of the native and recombinant catalytic subunit of human DNA polymerase gamma: identification of residues critical for exonuclease activity and dideoxynucleotide sensitivity. Biochemistry 37:10529–10539
Lim SE, Longley MJ, Copeland WC (1999) The mitochondrial p55 accessory subunit of human DNA polymerase gamma enhances DNA binding, promotes processive DNA synthesis, and confers N-ethylmaleimide resistance. J Biol Chem 274:38197–38203
Boosalis MS, Petruska J, Goodman MF (1987) DNA polymerase insertion fidelity. Gel assay for site-specific kinetics. J Biol Chem 262:14689–14696
Mendelman LV, Petruska J, Goodman MF (1990) Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. J Biol Chem 265:2338–2346
Chan SSL, Longley MJ, Copeland WC (2005) The common A467T mutation in the human mitochondrial DNA polymerase (POLG) compromises catalytic efficiency and interaction with the accessory subunit. J Biol Chem 280:31341–31346
Acknowledgments
This work was supported by NIH, NIEHS intramural research funds (ES 065078 and ES 065080).
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Copeland, W.C., Kasiviswanathan, R., Longley, M.J. (2016). Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ. In: McKenzie, M. (eds) Mitochondrial DNA. Methods in Molecular Biology, vol 1351. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3040-1_2
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DOI: https://doi.org/10.1007/978-1-4939-3040-1_2
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