Skip to main content
SpringerLink
Log in
Book cover

DNA Replication pp 169–186Cite as

Chromatin Immunoprecipitation to Detect DNA Replication and Repair Factors

  • Protocol

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1300))

Abstract

DNA replication is tightly coupled with DNA repair processes in order to preserve genomic integrity. During DNA replication, the replication fork encounters a variety of obstacles including DNA damage/adducts, secondary structures, and programmed fork-blocking sites, which are all difficult to replicate. The replication fork also collides with the transcription machinery, which shares the template DNA with the replisome complex. Under these conditions, replication forks stall, causing replication stress and/or fork collapse, ultimately leading to genomic instability. The mechanisms to overcome these replication problems remain elusive. Therefore, it is important to investigate how DNA repair and replication factors are recruited and coordinated at chromosomal regions that are difficult to replicate. In this chapter, we describe a chromatin immunoprecipitation method to locate proteins required for DNA repair during DNA replication in the fission yeast Schizosaccharomyces pombe. This method can also easily be adapted to study replisome components or chromatin-associated factors.

This is a preview of subscription content, 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   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
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

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Leman AR, Noguchi E (2013) The replication fork: understanding the eukaryotic replication machinery and the challenges to genome duplication. Genes 4:1–32

    Article  PubMed Central  PubMed  Google Scholar 

  2. Magdalou I, Lopez BS, Pasero P, Lambert SA (2014) The causes of replication stress and their consequences on genome stability and cell fate. Semin Cell Dev Biol 30:154–164

    Article  CAS  PubMed  Google Scholar 

  3. Zeman MK, Cimprich KA (2014) Causes and consequences of replication stress. Nat Cell Biol 16:2–9

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  4. Menck CF, Munford V (2014) DNA repair diseases: what do they tell us about cancer and aging? Genet Mol Biol 37:220–233

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Blow JJ (1993) Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor. J Cell Biol 122:993–1002

    Article  CAS  PubMed  Google Scholar 

  6. Blow JJ, Dutta A (2005) Preventing re-replication of chromosomal DNA. Nat Rev Mol Cell Biol 6:476–486

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Alver RC, Chadha GS, Blow JJ (2014) The contribution of dormant origins to genome stability: from cell biology to human genetics. DNA Repair (Amst) 19:182–189

    Article  CAS  Google Scholar 

  8. Nishitani H, Lygerou Z, Nishimoto T, Nurse P (2000) The Cdt1 protein is required to license DNA for replication in fission yeast. Nature 404:625–628

    Article  CAS  PubMed  Google Scholar 

  9. Saxena S, Dutta A (2005) Geminin-Cdt1 balance is critical for genetic stability. Mutat Res 569:111–121

    Article  CAS  PubMed  Google Scholar 

  10. Blow JJ, Gillespie PJ (2008) Replication licensing and cancer–a fatal entanglement? Nat Rev Cancer 8:799–806

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Mihaylov IS, Kondo T, Jones L, Ryzhikov S, Tanaka J, Zheng J, Higa LA, Minamino N, Cooley L, Zhang H (2002) Control of DNA replication and chromosome ploidy by geminin and cyclin A. Mol Cell Biol 22:1868–1880

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. San Filippo J, Sung P, Klein H (2008) Mechanism of eukaryotic homologous recombination. Annu Rev Biochem 77:229–257

    Article  CAS  PubMed  Google Scholar 

  13. Sung P, Trujillo KM, Van Komen S (2000) Recombination factors of Saccharomyces cerevisiae. Mutat Res 451:257–275

    Article  CAS  PubMed  Google Scholar 

  14. Game JC (1993) DNA double-strand breaks and the RAD50-RAD57 genes in Saccharomyces. Semin Cancer Biol 4:73–83

    CAS  PubMed  Google Scholar 

  15. Hays SL, Firmenich AA, Massey P, Banerjee R, Berg P (1998) Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA. Mol Cell Biol 18:4400–4406

    PubMed Central  CAS  PubMed  Google Scholar 

  16. Mortensen UH, Bendixen C, Sunjevaric I, Rothstein R (1996) DNA strand annealing is promoted by the yeast Rad52 protein. Proc Natl Acad Sci U S A 93:10729–10734

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Krejci L, Song B, Bussen W, Rothstein R, Mortensen UH, Sung P (2002) Interaction with Rad51 is indispensable for recombination mediator function of Rad52. J Biol Chem 277:40132–40141

    Article  CAS  PubMed  Google Scholar 

  18. Fabre F, Chan A, Heyer WD, Gangloff S (2002) Alternate pathways involving Sgs1/Top3, Mus81/ Mms4, and Srs2 prevent formation of toxic recombination intermediates from single-stranded gaps created by DNA replication. Proc Natl Acad Sci U S A 99:16887–16892

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  19. McEachern MJ, Haber JE (2006) Break-induced replication and recombinational telomere elongation in yeast. Annu Rev Biochem 75:111–135

    Article  CAS  PubMed  Google Scholar 

  20. Noguchi E, Noguchi C, Du LL, Russell P (2003) Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1. Mol Cell Biol 23:7861–7874

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Noguchi E, Noguchi C, McDonald WH, Yates JR 3rd, Russell P (2004) Swi1 and Swi3 are components of a replication fork protection complex in fission yeast. Mol Cell Biol 24:8342–8355

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Tourriere H, Versini G, Cordon-Preciado V, Alabert C, Pasero P (2005) Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53. Mol Cell 19:699–706

    Article  CAS  PubMed  Google Scholar 

  23. Urtishak KA, Smith KD, Chanoux RA, Greenberg RA, Johnson FB, Brown EJ (2009) Timeless maintains genomic stability and suppresses sister chromatid exchange during unperturbed DNA replication. J Biol Chem 284:8777–8785

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  24. Gilmour DS, Lis JT (1984) Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes. Proc Natl Acad Sci U S A 81:4275–4279

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  25. Gilmour DS, Lis JT (1985) In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster. Mol Cell Biol 5:2009–2018

    PubMed Central  CAS  PubMed  Google Scholar 

  26. Boyd KE, Farnham PJ (1997) Myc versus USF: discrimination at the cad gene is determined by core promoter elements. Mol Cell Biol 17:2529–2537

    PubMed Central  CAS  PubMed  Google Scholar 

  27. Rapp JB, Ansbach AB, Noguchi C, Noguchi E (2009) Chromatin immunoprecipitation of replication factors moving with the replication fork. Methods Mol Biol 521:191–202

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  28. Gadaleta MC, Iwasaki O, Noguchi C, Noma K, Noguchi E (2013) New vectors for epitope tagging and gene disruption in Schizosaccharomyces pombe. Biotechniques 55:257–263

    CAS  PubMed  Google Scholar 

  29. Southern JA, Young DF, Heaney F, Baumgartner WK, Randall RE (1991) Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics. J Gen Virol 72:1551–1557

    Article  CAS  PubMed  Google Scholar 

  30. Funakoshi M, Hochstrasser M (2009) Small epitope-linker modules for PCR-based C-terminal tagging in Saccharomyces cerevisiae. Yeast 26:185–192

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Fantes P (1979) Epistatic gene interactions in the control of division in fission yeast. Nature 279:428–430

    Article  CAS  PubMed  Google Scholar 

  32. Kurdistani SK, Grunstein M (2003) In vivo protein-protein and protein-DNA crosslinking for genomewide binding microarray. Methods 31:90–95

    Article  CAS  PubMed  Google Scholar 

  33. Cristea IM, Williams R, Chait BT, Rout MP (2005) Fluorescent proteins as proteomic probes. Mol Cell Proteomics 4:1933–1941

    Article  CAS  PubMed  Google Scholar 

  34. Vashist SK, Czilwik G, van Oordt T, von Stetten F, Zengerle R, Marion Schneider E, Luong JH (2014) One-step kinetics-based immunoassay for the highly sensitive detection of C-reactive protein in less than 30 min. Anal Biochem 456C:32–37

    Article  Google Scholar 

  35. Neurauter AA, Bonyhadi M, Lien E, Nokleby L, Ruud E, Camacho S, Aarvak T (2007) Cell isolation and expansion using Dynabeads. Adv Biochem Eng Biotechnol 106:41–73

    CAS  PubMed  Google Scholar 

  36. Nelson JD, Denisenko O, Sova P, Bomsztyk K (2006) Fast chromatin immunoprecipitation assay. Nucleic Acids Res 34:e2

    Article  PubMed Central  PubMed  Google Scholar 

  37. Moser BA, Subramanian L, Chang YT, Noguchi C, Noguchi E, Nakamura TM (2009) Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres. EMBO J 28:810–820

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Noguchi C, Rapp JB, Skorobogatko YV, Bailey LD, Noguchi E (2012) Swi1 associates with chromatin through the DDT domain and recruits Swi3 to preserve genomic integrity. PLoS One 7:e43988

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Roseaulin LC, Noguchi C, Martinez E, Ziegler MA, Toda T, Noguchi E (2013) Coordinated degradation of replisome components ensures genome stability upon replication stress in the absence of the replication fork protection complex. PLoS Genet 9:e1003213

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Kim HS, Vanoosthuyse V, Fillingham J, Roguev A, Watt S, Kislinger T, Treyer A, Carpenter LR, Bennett CS, Emili A, Greenblatt JF, Hardwick KG, Krogan NJ, Bahler J, Keogh MC (2009) An acetylated form of histone H2A.Z regulates chromosome architecture in Schizosaccharomyces pombe. Nat Struct Mol Biol 16:1286–1293

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  41. Gaillard H, Herrera-Moyano E, Aguilera A (2013) Transcription-associated genome instability. Chem Rev 113:8638–8661

    Article  CAS  PubMed  Google Scholar 

  42. Lin Y, Larson KL, Dorer R, Smith GR (1992) Meiotically induced rec7 and rec8 genes of Schizosaccharomyces pombe. Genetics 132:75–85

    PubMed Central  CAS  PubMed  Google Scholar 

  43. Kim N, Abdulovic AL, Gealy R, Lippert MJ, Jinks-Robertson S (2007) Transcription-associated mutagenesis in yeast is directly proportional to the level of gene expression and influenced by the direction of DNA replication. DNA Repair (Amst) 6:1285–1296

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by NIH grant (GM0776043 to E.N.).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 245 N 15th Street, Philadelphia, PA, 19102, USA

    Mariana C. Gadaleta, Chiaki Noguchi & Eishi Noguchi

  2. Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, PA, USA

    Osamu Iwasaki & Ken-Ichi Noma

Authors
  1. Mariana C. Gadaleta
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Osamu Iwasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chiaki Noguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ken-Ichi Noma
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eishi Noguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eishi Noguchi .

Editor information

Editors and Affiliations

  1. Coventry, United Kingdom

    Sonya Vengrova

  2. Warwick Medical School, University of Warwick, Coventry, United Kingdom

    Jacob Dalgaard

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Gadaleta, M.C., Iwasaki, O., Noguchi, C., Noma, KI., Noguchi, E. (2015). Chromatin Immunoprecipitation to Detect DNA Replication and Repair Factors. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 1300. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2596-4_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2596-4_12

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2595-7

  • Online ISBN: 978-1-4939-2596-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation