Assays of Bypass Replication of Genotoxic Lesions in Cell-Free Extracts

  • Nana Nikolaishvili-Feinberg
  • Marila Cordeiro-Stone
Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 920)

Abstract

The in vitro replication assay described here measures bidirectional replication of a circular double- stranded DNA template upon initiation at the SV40 origin. It models a single eukaryotic replication unit (replicon) and recapitulates the biochemical steps involved in the catalysis of both leading and lagging strand synthesis during semiconservative DNA replication. Except for the SV40 large T antigen, all other proteins necessary for initiation and assembly of functional replication forks are provided by the cell-free extract. This assay can be used to demonstrate bypass replication of genotoxic lesions. It supports replication across a specific damaged site on the template DNA (i.e., translesion synthesis) by specialized DNA polymerases. This chapter illustrates the efficient translesion synthesis of UV-induced thymine dimers by DNA polymerase eta.

Key words

Translesion synthesis Pyrimidine dimers Ultraviolet light DNA polymerase eta SV40 large T antigen DNA replication Human cells 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgement

This research was supported by the US Public Health Service Award CA55065 from the National Cancer Institute, National Institutes of Health. We thank Dr. Tadayoshi Bessho for assistance in preparing lesion-containing oligonucleotides and Dr. Stephen Chaney for access to HPLC equipment (Department of Biochemistry and Biophysics, UNC Chapel Hill). We are grateful to Dr. Thomas Kunkel (NIEHS) for the gift of M13mp2SV oriL and oriR. We thank Dr. John J. McNulty for reading the manuscript.

References

  1. 1.
    Unk I, Hajdu I, Blastyak A, Haracska L (2010) Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance. DNA Repair (Amst) 9:257–267CrossRefGoogle Scholar
  2. 2.
    Branzei D (2011) Ubiquitin family modifications and template switching. FEBS Lett. doi: 10.1016/j.febslet.2011.04.053
  3. 3.
    Livneh Z, Ziv O, Shachar S (2010) Multiple two-polymerase mechanisms in mammalian translesion DNA synthesis. Cell Cycle 9:729–735PubMedCrossRefGoogle Scholar
  4. 4.
    Lange SS, Takata K, Wood RD (2011) DNA polymerases and cancer. Nat Rev Cancer 11:96–110PubMedCrossRefGoogle Scholar
  5. 5.
    Shachar S, Ziv O, Avkin S, Adar S, Wittschieben J, Reissner T, Chaney S, Friedberg EC, Wang Z, Carell T, Geacintov N, Livneh Z (2009) Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals. EMBO J 28:383–393PubMedCrossRefGoogle Scholar
  6. 6.
    Avkin S, Goldsmith M, Velasco-Miguel S, Geacintov N, Friedberg EC, Livneh Z (2004) Quantitative analysis of translesion DNA synthesis across a benzo[a]pyrene-guanine adduct in mammalian cells: the role of DNA polymerase kappa. J Biol Chem 279:53298–53305PubMedCrossRefGoogle Scholar
  7. 7.
    Waga S, Stillman B (1998) The DNA replication fork in eukaryotic cells. Annu Rev Biochem 67:721–751PubMedCrossRefGoogle Scholar
  8. 8.
    Thomas DC, Veaute X, Kunkel TA, Fuchs RP (1994) Mutagenic replication in human cell extracts of DNA containing site-specific N-2-acetylaminofluorene adducts. Proc Natl Acad Sci U S A 91:7752–7756PubMedCrossRefGoogle Scholar
  9. 9.
    Thomas DC, Veaute X, Fuchs RP, Kunkel TA (1995) Frequency and fidelity of translesion synthesis of site-specific N-2-acetylaminofluorene adducts during DNA replication in a human cell extract. J Biol Chem 270:21226–21233PubMedCrossRefGoogle Scholar
  10. 10.
    Carty MP, Lawrence CW, Dixon K (1996) Complete replication of plasmid DNA containing a single UV-induced lesion in human cell extracts. J Biol Chem 271:9637–9647PubMedCrossRefGoogle Scholar
  11. 11.
    Svoboda DL, Vos JM (1995) Differential replication of a single, UV-induced lesion in the leading or lagging strand by a human cell extract: fork uncoupling or gap formation. Proc Natl Acad Sci U S A 92:11975–11979PubMedCrossRefGoogle Scholar
  12. 12.
    Cordeiro-Stone M, Zaritskaya LS, Price LK, Kaufmann WK (1997) Replication fork bypass of a pyrimidine dimer blocking leading strand DNA synthesis. J Biol Chem 272:13945–13954PubMedCrossRefGoogle Scholar
  13. 13.
    Nikolaishvili-Feinberg N, Jenkins GS, Nevis KR, Staus DP, Scarlett CO, Unsal-Kacmaz K, Kaufmann WK, Cordeiro-Stone M (2008) Ubiquitylation of proliferating cell nuclear antigen and recruitment of human DNA polymerase eta. Biochemistry 47:4141–4150PubMedCrossRefGoogle Scholar
  14. 14.
    Masutani C, Araki M, Yamada A, Kusumoto R, Nogimori T, Maekawa T, Iwai S, Hanaoka F (1999) Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity. EMBO J 18:3491–3501PubMedCrossRefGoogle Scholar
  15. 15.
    Nikolaishvili-Feinberg N, Cordeiro-Stone M (2001) Bypass replication in vitro of UV-induced photoproducts blocking leading or lagging strand synthesis. Biochemistry 40:15215–15223PubMedCrossRefGoogle Scholar
  16. 16.
    Zhao X, Kao JL, Taylor JS (1995) Preparation and characterization of a deoxyoligonucleotide 49-mer containing a site-specific thymidylyl-(3′,5′)-deoxyadenosine photoproduct. Biochemistry 34:1386–1392PubMedCrossRefGoogle Scholar
  17. 17.
    Roberts JD, Kunkel TA (1988) Fidelity of a human cell DNA replication complex. Proc Natl Acad Sci U S A 85:7064–7068PubMedCrossRefGoogle Scholar
  18. 18.
    Roberts JD, Thomas DC, Kunkel TA (1991) Exonucleolytic proofreading of leading and lagging strand DNA replication errors. Proc Natl Acad Sci U S A 88:3465–3469PubMedCrossRefGoogle Scholar
  19. 19.
    Roberts JD, Izuta S, Thomas DC, Kunkel TA (1994) Mispair-, site-, and strand-specific error rates during simian virus 40 origin-dependent replication in vitro with excess deoxythymidine triphosphate. J Biol Chem 269:1711–1717PubMedGoogle Scholar
  20. 20.
    Li JJ, Kelly TJ (1984) Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A 81:6973–6977PubMedCrossRefGoogle Scholar
  21. 21.
    Li JJ, Kelly TJ (1985) Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication. Mol Cell Biol 5:1238–1246PubMedGoogle Scholar
  22. 22.
    Roberts JD, Kunkel TA (1993) Fidelity of DNA replication in human cells. Methods Mol Genet 2:295–313Google Scholar
  23. 23.
    Smith CA, Taylor JS (1993) Preparation and characterization of a set of deoxyoligonucleotide 49-mers containing site-specific cis-syn, trans-syn-I, (6–4), and Dewar photoproducts of thymidylyl(3′–>5′)-thymidine. J Biol Chem 268:11143–11151PubMedGoogle Scholar
  24. 24.
    Brown TA (2010) Gene cloning and DNA analysis: an introduction. Wiley-Blackwell, ChichesterGoogle Scholar
  25. 25.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual cold spring harbor laboratory press. Cold Spring Harbor, New YorkGoogle Scholar
  26. 26.
    Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492PubMedCrossRefGoogle Scholar
  27. 27.
    Kunkel TA, Bebenek K, McClary J (1991) Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol 204:125–139PubMedCrossRefGoogle Scholar
  28. 28.
    Stillman BW, Gluzman Y (1985) Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol 5:2051–2060PubMedGoogle Scholar
  29. 29.
    Stillman B (1986) Chromatin assembly during SV40 DNA replication in vitro. Cell 45:555–565PubMedCrossRefGoogle Scholar
  30. 30.
    Friedberg EC, Walker GC, Siede W (1995) DNA repair and mutagenesis. ASM Press, Washington, D.C, pp 1–58Google Scholar
  31. 31.
    Wobbe CR, Dean FB, Murakami Y, Weissbach L, Hurwitz J (1986) Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains. Proc Natl Acad Sci U S A 83:4612–4616PubMedCrossRefGoogle Scholar
  32. 32.
    Cordeiro-Stone M, Nikolaishvili-Feinberg N (2002) Asymmetry of DNA replication and translesion synthesis of UV-induced thymine dimers. Mutat Res 510:91–106PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Nana Nikolaishvili-Feinberg
    • 1
  • Marila Cordeiro-Stone
    • 2
    • 3
  1. 1.Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center and Center for Environmental Health and SusceptibilityUniversity of North Carolina at Chapel HillChapel HillUSA
  2. 2.Department of Pathology and Laboratory MedicineLineberger Comprehensive Cancer Center University of North Carolina at Chapel HillChapel HillUSA
  3. 3.Center for Environmental Health and SusceptibilityUniversity of North Carolina at Chapel HillChapel HillUSA

Personalised recommendations