Skip to main content
SpringerLink
Log in

Characterizing Complex Nucleic Acid Interactions of LINE1 ORF1p by Single Molecule Force Spectroscopy

  • Protocol
  • First Online:
RNA Chaperones

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2106))

Abstract

The L1 retrotransposon is the dominant transposable element in mammalian genomes. L1 comprises at least 20% of the human genome. While most L1 regions are inactive, a few still retain the ability to retrotranspose. L1 encodes two proteins, ORF1p and ORF2p, which are required for retrotransposition. During retrotransposition, ORF2p functions as the reverse transcriptase and the endonuclease. ORF1p is a nucleic acid chaperone that binds nucleic acids with high affinity. However, to date, a detailed mechanistic understanding of ORF1p function in L1 retrotransposition is lacking. The single molecule DNA stretching methods described here have been extensively used to understand ORF1p’s complex nucleic acid binding properties. By correlating these properties to ORF1p’s ability to support L1 retrotransposition in in vivo cell-culture based assays, these studies have significantly contributed to advance the understanding of ORF1p function. Although described in the context of ORF1p, these methods provide a general mechanism to study complex protein-DNA interactions.

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 109.00
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 199.99
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. Furano AV (2000) The biological properties and evolutionary dynamics of mammalian LINE-1 retrotransposons. Prog Nucleic Acid Res Mol Biol 64:255–294

    CAS  PubMed  Google Scholar 

  2. Lander ES, Linton LM, Birren B et al (2001) Initial sequencing and analysis of the human genome. Nature 409(6822):860–921

    CAS  PubMed  Google Scholar 

  3. Kazazian HH Jr, Moran JV (2017) Mobile DNA in health and disease. N Engl J Med 377(4):361–370

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Luan DD, Korman MH, Jakubczak JL et al (1993) Reverse transcription of R2Bm RNA is primed by a nick at the chromosomal target site: a mechanism for non-LTR retrotransposition. Cell 72(4):595–605

    CAS  PubMed  Google Scholar 

  5. Martin SL, Bushman FD (2001) Nucleic acid chaperone activity of the ORF1 protein from the mouse LINE-1 retrotransposon. Mol Cell Biol 21(2):467–475

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Esnault C, Maestre J, Heidmann T (2000) Human LINE retrotransposons generate processed pseudogenes. Nat Genet 24(4):363–367

    CAS  PubMed  Google Scholar 

  7. Wei W, Gilbert N, Ooi SL et al (2001) Human L1 retrotransposition: cis preference versus trans complementation. Mol Cell Biol 21(4):1429–1439

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Boissinot S, Furano AV (2001) Adaptive evolution in LINE-1 retrotransposons. Mol Biol Evol 18(12):2186–2194

    CAS  PubMed  Google Scholar 

  9. Boissinot S, Davis J, Entezam A et al (2006) Fitness cost of LINE-1 (L1) activity in humans. Proc Natl Acad Sci U S A 103(25):9590–9594

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Myers S, Bottolo L, Freeman C et al (2005) A fine-scale map of recombination rates and hotspots across the human genome. Science 310(5746):321–324

    CAS  PubMed  Google Scholar 

  11. Bourc'his D, Bestor TH (2004) Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature 431(7004):96–99

    CAS  PubMed  Google Scholar 

  12. Soper SFC, van der Heijden GW, Hardiman TC et al (2008) Mouse maelstrom, a component of nuage, is essential for spermatogenesis and transposon repression in meiosis. Dev Cell 15(2):285–297

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Skowronski J, Fanning TG, Singer MF (1988) Unit-length line-1 transcripts in human teratocarcinoma cells. Mol Cell Biol 8(4):1385–1397

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Boissinot S, Chevret P, Furano AV (2000) L1 (LINE-1) retrotransposon evolution and amplification in recent human history. Mol Biol Evol 17(6):915–928

    CAS  PubMed  Google Scholar 

  15. Boissinot S, Entezam A, Young L et al (2004) The insertional history of an active family of L1 retrotransposons in humans. Genome Res 14(7):1221–1231

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Chen JM, Stenson PD, Cooper DN et al (2005) A systematic analysis of LINE-1 endonuclease-dependent retrotranspositional events causing human genetic disease. Hum Genet 117(5):411–427

    CAS  PubMed  Google Scholar 

  17. Beck CR, Garcia-Perez JL, Badge RM et al (2011) LINE-1 elements in structural variation and disease. Annu Rev Genomics Hum Genet 12:187–215

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Huang CR, Schneider AM, Lu Y et al (2010) Mobile interspersed repeats are major structural variants in the human genome. Cell 141(7):1171–1182

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Iskow RC, McCabe MT, Mills RE et al (2010) Natural mutagenesis of human genomes by endogenous retrotransposons. Cell 141(7):1253–1261

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Moran JV, Holmes SE, Naas TP et al (1996) High frequency retrotransposition in cultured mammalian cells. Cell 87(5):917–927

    CAS  PubMed  Google Scholar 

  21. Doucet AJ, Hulme AE, Sahinovic E et al (2010) Characterization of LINE-1 ribonucleoprotein particles. PLoS Genet 6(10):e1001150

    PubMed  PubMed Central  Google Scholar 

  22. Kulpa DA, Moran JV (2006) Cis-preferential LINE-1 reverse transcriptase activity in ribonucleoprotein particles. Nat Struct Mol Biol 13(7):655–660

    CAS  PubMed  Google Scholar 

  23. Kulpa DA, Moran JV (2005) Ribonucleoprotein particle formation is necessary but not sufficient for LINE-1 retrotransposition. Hum Mol Genet 14(21):3237–3248

    CAS  PubMed  Google Scholar 

  24. Martin SL (1991) Ribonucleoprotein particles with LINE-1 RNA in mouse embryonal carcinoma cells. Mol Cell Biol 11(9):4804–4807

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Feng Q, Moran JV, Kazazian HH Jr et al (1996) Human L1 retrotransposon encodes a conserved endonuclease required for retrotransposition. Cell 87(5):905–916

    CAS  PubMed  Google Scholar 

  26. Mathias SL, Scott AF, Kazazian HHJ et al (1991) Reverse transcriptase encoded by a human transposable element. Science 254(5039):1808–1810

    CAS  PubMed  Google Scholar 

  27. Eickbush TH, Malik HS (2002) Origins and evolution of retrotransposons. In: Craig NL, Craigie R, Gellert M, Lambowitz AM (eds) Mobile DNA II. American Society of Microbiology Press, Washington, DC, pp 1111–1144

    Google Scholar 

  28. Basame S, Wai-lun Li P, Howard G et al (2006) Spatial assembly and RNA binding stoichiometry of a LINE-1 protein essential for retrotransposition. J Mol Biol 357(2):351–357

    CAS  PubMed  Google Scholar 

  29. Khazina E, Truffault V, Buttner R et al (2011) Trimeric structure and flexibility of the L1ORF1 protein in human L1 retrotransposition. Nat Struct Mol Biol 18(9):1006–1014

    CAS  PubMed  Google Scholar 

  30. Khazina E, Weichenrieder O (2009) Non-LTR retrotransposons encode noncanonical RRM domains in their first open reading frame. Proc Natl Acad Sci U S A 106(3):731–736

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Martin SL, Branciforte D, Keller D et al (2003) Trimeric structure for an essential protein in L1 retrotransposition. Proc Natl Acad Sci U S A 100(24):13815–13820

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Januszyk K, Li PW, Villareal V et al (2007) Identification and solution structure of a highly conserved C-terminal domain within ORF1p required for retrotransposition of long interspersed nuclear element-1. J Biol Chem 282(34):24893–24904

    CAS  PubMed  Google Scholar 

  33. Naufer MN, Furano AV, Williams MC (2018) Protein-nucleic acid interactions of LINE-1 ORF1p. Semin Cell Dev Biol 86:140–149

    PubMed  PubMed Central  Google Scholar 

  34. Martin SL, Cruceanu M, Branciforte D et al (2005) LINE-1 retrotransposition requires the nucleic acid chaperone activity of the ORF1 protein. J Mol Biol 348(3):549–561

    CAS  PubMed  Google Scholar 

  35. Evans JD, Peddigari S, Chaurasiya KR et al (2011) Paired mutations abolish and restore the balanced annealing and melting activities of ORF1p that are required for LINE-1 retrotransposition. Nucleic Acids Res 39(13):5611–5621

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Martin SL, Bushman D, Wang F et al (2008) A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity. Nucleic Acids Res 36(18):5845–5854

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Naufer MN, Callahan KE, Cook PR et al (2016) L1 retrotransposition requires rapid ORF1p oligomerization, a novel coiled coil-dependent property conserved despite extensive remodeling. Nucleic Acids Res 44(1):281–293

    CAS  PubMed  Google Scholar 

  38. Chaurasiya KR, Paramanathan T, McCauley MJ et al (2010) Biophysical characterization of DNA binding from single molecule force measurements. Phys Life Rev 7(3):299–341

    PubMed  PubMed Central  Google Scholar 

  39. Williams MC, Wenner JR, Rouzina I et al (2001) Effect of pH on the overstretching transition of double-stranded DNA: evidence of force-induced DNA melting. Biophys J 80(2):874–881

    CAS  PubMed  PubMed Central  Google Scholar 

  40. McCauley MJ, Williams MC (2009) Optical tweezers experiments resolve distinct modes of DNA-protein binding. Biopolymers 91(4):265–282

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Cruceanu M, Urbaneja MA, Hixson CV et al (2006) Nucleic acid binding and chaperone properties of HIV-1 gag and nucleocapsid proteins. Nucleic Acids Res 34(2):593–605

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Callahan KE, Hickman AB, Jones CE et al (2012) Polymerization and nucleic acid-binding properties of human L1 ORF1 protein. Nucleic Acids Res 40(2):813–827

    CAS  PubMed  Google Scholar 

  43. Chaurasiya KR, McCauley MJ, Wang W et al (2014) Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein. Nat Chem 6(1):28–33

    CAS  PubMed  Google Scholar 

  44. Ibarra B, Chemla YR, Plyasunov S et al (2009) Proofreading dynamics of a processive DNA polymerase. EMBO J 28(18):2794–2802

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Wuite GJ, Smith SB, Young M et al (2000) Single-molecule studies of the effect of template tension on T7 DNA polymerase activity. Nature 404(6773):103–106

    CAS  PubMed  Google Scholar 

  46. Naufer MN, Murison DA, Rouzina I et al (2017) Single-molecule mechanochemical characterization of E. coli pol III core catalytic activity. Protein Sci 26(7):1413–1426

    CAS  PubMed  PubMed Central  Google Scholar 

  47. King GA, Gross P, Bockelmann U et al (2013) Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy. Proc Natl Acad Sci U S A 110(10):3859–3864

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhang X, Chen H, Le S et al (2013) Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching by single-molecule calorimetry. Proc Natl Acad Sci U S A 110(10):3865–3870

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Williams MC, Gorelick RJ, Musier-Forsyth K (2002) Specific zinc-finger architecture required for HIV-1 nucleocapsid protein's nucleic acid chaperone function. Proc Natl Acad Sci U S A 99(13):8614–8619

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Williams MC, Rouzina I, Wenner JR et al (2001) Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching. Proc Natl Acad Sci U S A 98(11):6121–6126

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Williams MC, Rouzina I, Bloomfield VA (2002) Thermodynamics of DNA interactions from single molecule stretching experiments. Acc Chem Res 35(3):159–166

    CAS  PubMed  Google Scholar 

  52. Wu H, Rouzina I, Williams MC (2010) Single-molecule stretching studies of RNA chaperones. RNA Biol 7(6):712–723

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Cruceanu M, Gorelick RJ, Musier-Forsyth K et al (2006) Rapid kinetics of protein-nucleic acid interaction is a major component of HIV-1 nucleocapsid protein's nucleic acid chaperone function. J Mol Biol 363(5):867–877

    CAS  PubMed  Google Scholar 

  54. Levin JG, Guo J, Rouzina I et al (2005) Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. Prog Nucleic Acid Res Mol Biol 80:217–286

    CAS  PubMed  Google Scholar 

  55. Post K, Olson ED, Naufer MN et al (2016) Mechanistic differences between HIV-1 and SIV nucleocapsid proteins and cross-species HIV-1 genomic RNA recognition. Retrovirology 13(1):89

    PubMed  PubMed Central  Google Scholar 

  56. Odijk T (1995) Stiff chains and filaments under tension. Macromolecules 28(20):7016–7018

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Physics, Northeastern University, Boston, MA, USA

    M. Nabuan Naufer & Mark C. Williams

Authors
  1. M. Nabuan Naufer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark C. Williams
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark C. Williams .

Editor information

Editors and Affiliations

  1. Department of Pediatric Hematology, Oncology and Stem Cell Transplantation, University Hospital of Regensburg, Regensburg, Germany

    Tilman Heise

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Naufer, M.N., Williams, M.C. (2020). Characterizing Complex Nucleic Acid Interactions of LINE1 ORF1p by Single Molecule Force Spectroscopy. In: Heise, T. (eds) RNA Chaperones. Methods in Molecular Biology, vol 2106. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0231-7_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0231-7_18

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0230-0

  • Online ISBN: 978-1-0716-0231-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation