Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
DNA Repair Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 920))

  • 6922 Accesses

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  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) 9:257–267

    Article  CAS  Google Scholar 

  2. Branzei D (2011) Ubiquitin family modifications and template switching. FEBS Lett. doi:10.1016/j.febslet.2011.04.053

  3. Livneh Z, Ziv O, Shachar S (2010) Multiple two-polymerase mechanisms in mammalian translesion DNA synthesis. Cell Cycle 9:729–735

    Article  PubMed  CAS  Google Scholar 

  4. Lange SS, Takata K, Wood RD (2011) DNA polymerases and cancer. Nat Rev Cancer 11:96–110

    Article  PubMed  CAS  Google Scholar 

  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 28:383–393

    Article  PubMed  CAS  Google Scholar 

  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 279:53298–53305

    Article  PubMed  CAS  Google Scholar 

  7. Waga S, Stillman B (1998) The DNA replication fork in eukaryotic cells. Annu Rev Biochem 67:721–751

    Article  PubMed  CAS  Google Scholar 

  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 91:7752–7756

    Article  PubMed  CAS  Google Scholar 

  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 270:21226–21233

    Article  PubMed  CAS  Google Scholar 

  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 271:9637–9647

    Article  PubMed  CAS  Google Scholar 

  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 92:11975–11979

    Article  PubMed  CAS  Google Scholar 

  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 272:13945–13954

    Article  PubMed  CAS  Google Scholar 

  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 47:4141–4150

    Article  PubMed  CAS  Google Scholar 

  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 18:3491–3501

    Article  PubMed  CAS  Google Scholar 

  15. Nikolaishvili-Feinberg N, Cordeiro-Stone M (2001) Bypass replication in vitro of UV-induced photoproducts blocking leading or lagging strand synthesis. Biochemistry 40:15215–15223

    Article  PubMed  CAS  Google Scholar 

  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 34:1386–1392

    Article  PubMed  CAS  Google Scholar 

  17. Roberts JD, Kunkel TA (1988) Fidelity of a human cell DNA replication complex. Proc Natl Acad Sci U S A 85:7064–7068

    Article  PubMed  CAS  Google Scholar 

  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 88:3465–3469

    Article  PubMed  CAS  Google Scholar 

  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 269:1711–1717

    PubMed  CAS  Google Scholar 

  20. Li JJ, Kelly TJ (1984) Simian virus 40 DNA replication in vitro. Proc Natl Acad Sci U S A 81:6973–6977

    Article  PubMed  CAS  Google Scholar 

  21. Li JJ, Kelly TJ (1985) Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication. Mol Cell Biol 5:1238–1246

    PubMed  CAS  Google Scholar 

  22. Roberts JD, Kunkel TA (1993) Fidelity of DNA replication in human cells. Methods Mol Genet 2:295–313

    CAS  Google Scholar 

  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 268:11143–11151

    PubMed  CAS  Google Scholar 

  24. Brown TA (2010) Gene cloning and DNA analysis: an introduction. Wiley-Blackwell, Chichester

    Google Scholar 

  25. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual cold spring harbor laboratory press. Cold Spring Harbor, New York

    Google Scholar 

  26. Kunkel TA (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A 82:488–492

    Article  PubMed  CAS  Google Scholar 

  27. Kunkel TA, Bebenek K, McClary J (1991) Efficient site-directed mutagenesis using uracil-containing DNA. Methods Enzymol 204:125–139

    Article  PubMed  CAS  Google Scholar 

  28. Stillman BW, Gluzman Y (1985) Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells. Mol Cell Biol 5:2051–2060

    PubMed  CAS  Google Scholar 

  29. Stillman B (1986) Chromatin assembly during SV40 DNA replication in vitro. Cell 45:555–565

    Article  PubMed  CAS  Google Scholar 

  30. Friedberg EC, Walker GC, Siede W (1995) DNA repair and mutagenesis. ASM Press, Washington, D.C, pp 1–58

    Google Scholar 

  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 83:4612–4616

    Article  PubMed  CAS  Google Scholar 

  32. Cordeiro-Stone M, Nikolaishvili-Feinberg N (2002) Asymmetry of DNA replication and translesion synthesis of UV-induced thymine dimers. Mutat Res 510:91–106

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center and Center for Environmental Health and Susceptibility, University of North Carolina at Chapel Hill, Chapel Hill, USA

    Nana Nikolaishvili-Feinberg

  2. Department of Pathology and Laboratory Medicine, Lineberger Comprehensive Cancer Center University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Marila Cordeiro-Stone

  3. Center for Environmental Health and Susceptibility, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Marila Cordeiro-Stone

Authors
  1. Nana Nikolaishvili-Feinberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marila Cordeiro-Stone
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marila Cordeiro-Stone .

Editor information

Editors and Affiliations

  1. , Department of Molecular Biology, Aarhus University, C.F Møllers Allé bygning 1130, Århus C, 8000, Denmark

    Lotte Bjergbæk

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media New York

About this protocol

Cite this protocol

Nikolaishvili-Feinberg, N., Cordeiro-Stone, M. (2012). Assays of Bypass Replication of Genotoxic Lesions in Cell-Free Extracts. In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_34

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-998-3_34

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-997-6

  • Online ISBN: 978-1-61779-998-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation