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Analysis of Translesion DNA Synthesis by the Mitochondrial DNA Polymerase γ

  • William C. CopelandEmail author
  • Rajesh Kasiviswanathan
  • Matthew J. Longley
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)

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.

Key words

DNA polymerase γ Mitochondrial DNA polymerase DNA replication Translesion synthesis DNA repair Enzyme assays POLG 

Notes

Acknowledgments

This work was supported by NIH, NIEHS intramural research funds (ES 065078 and ES 065080).

References

  1. 1.
    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 108:13534–13539CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Cline SD (2012) Mitochondrial DNA damage and its consequences for mitochondrial gene expression. Biochim Biophys Acta 1819:979–991CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Copeland WC, Longley MJ (2014) Mitochondrial genome maintenance in health and disease. DNA Repair (Amst) 19:190–198CrossRefGoogle Scholar
  4. 4.
    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 277:18257–18265CrossRefPubMedGoogle Scholar
  5. 5.
    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 278:784–790CrossRefPubMedGoogle Scholar
  6. 6.
    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 24:5687–5693CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    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 24:6900–6906CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    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 25:8748–8754CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    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 37:2830–2840CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    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 428:97–100CrossRefPubMedGoogle Scholar
  11. 11.
    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 38:3233–3244CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    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) 10:826–834CrossRefPubMedCentralGoogle Scholar
  13. 13.
    Graziewicz MA, Longley MJ, Copeland WC (2006) DNA polymerase gamma in mitochondrial DNA replication and repair. Chem Rev 106:383–405CrossRefPubMedGoogle Scholar
  14. 14.
    Lee YS, Kennedy WD, Yin YW (2009) Structural insight into processive human mitochondrial DNA synthesis and disease-related polymerase mutations. Cell 139:312–324CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    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 285:1490–1499CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    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 16:2729–2739Google Scholar
  17. 17.
    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 32:397–405CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    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 287:9222–9229CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    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 288:14247–14255CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kasiviswanathan R, Longley MJ, Young MJ, Copeland WC (2010) Purification and functional characterization of human mitochondrial DNA polymerase gamma harboring disease mutations. Methods 51:379–384CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    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 329:45–57CrossRefPubMedGoogle Scholar
  22. 22.
    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 37:10529–10539CrossRefPubMedGoogle Scholar
  23. 23.
    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 274:38197–38203CrossRefPubMedGoogle Scholar
  24. 24.
    Boosalis MS, Petruska J, Goodman MF (1987) DNA polymerase insertion fidelity. Gel assay for site-specific kinetics. J Biol Chem 262:14689–14696PubMedGoogle Scholar
  25. 25.
    Mendelman LV, Petruska J, Goodman MF (1990) Base mispair extension kinetics. Comparison of DNA polymerase alpha and reverse transcriptase. J Biol Chem 265:2338–2346PubMedGoogle Scholar
  26. 26.
    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 280:31341–31346CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • William C. Copeland
    • 1
    Email author
  • Rajesh Kasiviswanathan
    • 1
  • Matthew J. Longley
    • 1
  1. 1.Mitochondrial DNA Replication Group, Genome Integrity and Structural Biology LaboratoryNational Institute of Environmental Health Sciences, National Institutes of HealthResearch Triangle ParkUSA

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