Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Guide-independent DNA cleavage by archaeal Argonaute from Methanocaldococcus jannaschii

Nature Microbiology volume 2, Article number: 17034 (2017) Cite this article

Subjects

Abstract

Prokaryotic Argonaute proteins acquire guide strands derived from invading or mobile genetic elements, via an unknown pathway, to direct guide-dependent cleavage of foreign DNA. Here, we report that Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action: the canonical guide-dependent endonuclease activity and a non-guided DNA endonuclease activity. The latter allows MjAgo to process long double-stranded DNAs, including circular plasmid DNAs and genomic DNAs. Degradation of substrates in a guide-independent fashion primes MjAgo for subsequent rounds of DNA cleavage. Chromatinized genomic DNA is resistant to MjAgo degradation, and recombinant histones protect DNA from cleavage in vitro. Mutational analysis shows that key residues important for guide-dependent target processing are also involved in guide-independent MjAgo function. This is the first characterization of guide-independent cleavage activity for an Argonaute protein potentially serving as a guide biogenesis pathway in a prokaryotic system.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Guide-directed target cleavage activity of MjAgo using canonical and non-canonical substrates.
Figure 2: MjAgo processes long linear and circular dsDNAs and genomic DNA in the absence of a guide DNA.
Figure 3: Characterization of DNA degradation products and influence on MjAgo-mediated plasmid degradation.
Figure 4: Mutational analysis of MjAgo guide-independent plasmid cleavage activity.
Figure 5: Putative model of guide-dependent and guide-independent DNA silencing by MjAgo.

Similar content being viewed by others

References

  1. Willkomm, S., Zander, A., Gust, A. & Grohmann, D. A prokaryotic twist on Argonaute function. Life 5, 538–553 (2015).

    Article  CAS  Google Scholar 

  2. Swarts, D. C. et al. The evolutionary journey of Argonaute proteins. Nat. Struct. Mol. Biol. 21, 743–753 (2014).

    Article  CAS  Google Scholar 

  3. Meister, G. Argonaute proteins: functional insights and emerging roles. Nat. Rev. Genet. 14, 447–459 (2013).

    Article  CAS  Google Scholar 

  4. Elbashir, S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).

    Article  CAS  Google Scholar 

  5. Elbashir, S. M., Lendeckel, W. & Tuschl, T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 15, 188–200 (2001).

    Article  CAS  Google Scholar 

  6. Zamore, P. D., Tuschl, T., Sharp, P. A. & Bartel, D. P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33 (2000).

    Article  CAS  Google Scholar 

  7. Swarts, D. C. et al. Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA. Nucleic Acids Res. 43, 5120–5129 (2015).

    Article  CAS  Google Scholar 

  8. Swarts, D. C. et al. DNA-guided DNA interference by a prokaryotic Argonaute. Nature 507, 258–261 (2014).

    Article  CAS  Google Scholar 

  9. Wang, Y. et al. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature 456, 921–926 (2008).

    Article  CAS  Google Scholar 

  10. Wang, Y. et al. Nucleation, propagation and cleavage of target RNAs in Ago silencing complexes. Nature 461, 754–761 (2009).

    Article  CAS  Google Scholar 

  11. Zander, A., Holzmeister, P., Klose, D., Tinnefeld, P. & Grohmann, D. Single-molecule FRET supports the two-state model of argonaute action. RNA Biol. 11, 45–56 (2014).

    Article  CAS  Google Scholar 

  12. Gao, F., Shen, X. Z., Jiang, F., Wu, Y. & Han, C. DNA-guided genome editing using the Natronobacterium gregoryi Argonaute. Nat. Biotechnol. 34, 768–773 (2016).

    Article  CAS  Google Scholar 

  13. Willkomm, S., Zander, A., Grohmann, D. & Restle, T. Mechanistic insights into archaeal and human argonaute substrate binding and cleavage properties. PLoS ONE 11, e0164695 (2016).

    Article  Google Scholar 

  14. Ma, J. B. et al. Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein. Nature 434, 666–670 (2005).

    Article  CAS  Google Scholar 

  15. Schirle, N. T. & MacRae, I. J. The crystal structure of human Argonaute2. Science 336, 1037–1040 (2012).

    Article  CAS  Google Scholar 

  16. Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D. J. Structure of the guide-strand-containing Argonaute silencing complex. Nature 456, 209–213 (2008).

    Article  CAS  Google Scholar 

  17. Frank, F., Sonenberg, N. & Nagar, B. Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2. Nature 465, 818–822 (2010).

    Article  CAS  Google Scholar 

  18. Nakanishi, K., Weinberg, D. E., Bartel, D. P. & Patel, D. J. Structure of yeast Argonaute with guide RNA. Nature 486, 368–374 (2012).

    Article  CAS  Google Scholar 

  19. Kaya, E. et al. A bacterial Argonaute with noncanonical guide RNA specificity. Proc. Natl Acad. Sci. USA 113, 4057–4062 (2016).

    Article  CAS  Google Scholar 

  20. Olovnikov, I., Chan, K., Sachidanandam, R., Newman, D. K. & Aravin, A. A. Bacterial Argonaute samples the transcriptome to identify foreign DNA. Mol. Cell 51, 594–605 (2013).

    Article  CAS  Google Scholar 

  21. Miyoshi, T., Ito, K., Murakami, R. & Uchiumi, T. Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute. Nat. Commun. 7, 11846 (2016).

    Article  CAS  Google Scholar 

  22. Blow, M. J. et al. The epigenomic landscape of prokaryotes. PLoS Genet. 12, e1005854 (2016).

  23. Loenen, W. A., Dryden, D. T., Raleigh, E. A., Wilson, G. G. & Murray, N. E. Highlights of the DNA cutters: a short history of the restriction enzymes. Nucleic Acids Res. 42, 3–19 (2014).

    Article  CAS  Google Scholar 

  24. Willkomm, S . et al. Structural and mechanistic insights into an archaeal DNA-guided Argonaute protein. Nat. Microbiol. 2, 17035 (2017).

    Article  CAS  Google Scholar 

  25. Elkayam, E. et al. The structure of human Argonaute-2 in complex with miR-20a. Cell 150, 100–110 (2012).

    Article  CAS  Google Scholar 

  26. Peeters, E., Driessen, R. P., Werner, F. & Dame, R. T. The interplay between nucleoid organization and transcription in archaeal genomes. Nat. Rev. Microbiol. 13, 333–341 (2015).

    Article  CAS  Google Scholar 

  27. Cheloufi, S., Dos Santos, C. O., Chong, M. M. & Hannon, G. J. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 465, 584–589 (2010).

    Article  CAS  Google Scholar 

  28. Chandradoss, S. D., Schirle, N. T., Szczepaniak, M., MacRae, I. J. & Joo, C. A dynamic search process underlies microRNA targeting. Cell 162, 96–107 (2015).

    Article  CAS  Google Scholar 

  29. Schirle, N. T., Sheu-Gruttadauria, J. & MacRae, I. J. Gene regulation. Structural basis for microRNA targeting. Science 346, 608–613 (2014).

    Article  CAS  Google Scholar 

  30. Parker, J. S. How to slice: snapshots of Argonaute in action. Silence 1, 3 (2010).

    Article  Google Scholar 

  31. Wagner, M. et al. Expanding and understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus. Biochem. Soc. Trans. 37, 97–101 (2009).

    Article  CAS  Google Scholar 

  32. Brock, T. D., Brock, K. M., Belly, R. T. & Weiss, R. L. Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch. Mikrobiol. 84, 54–68 (1972).

    Article  CAS  Google Scholar 

  33. Berkner, S., Wlodkowski, A., Albers, S. V. & Lipps, G. Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius. Extremophiles 14, 249–259 (2010).

    Article  CAS  Google Scholar 

  34. Wagner, M. et al. Versatile genetic tool box for the crenarchaeote Sulfolobus acidocaldarius. Front. Microbiol. 3, 214 (2012).

    Article  Google Scholar 

  35. Rachel, R. et al. Analysis of the ultrastructure of archaea by electron microscopy. Methods Cell Biol. 96, 47–69 (2010).

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank all members of the Grohmann laboratory and in particular K. Kramm for cloning of the MjAgo catalytic mutant. The authors thank G. Meister for discussions. Work in the Grohmann RNAP laboratory was funded by the Deutsche Forschungsgemeinschaft (GR 3840/2-1). S.V.A. and M.v.W. acknowledge funding by the European Research Council (starting grant ARCHAELLUM 311523). S.Sc. acknowledges funding by the Deutsche Forschungsgemeinschaft, the excellence cluster CIPSM and Fonds der Chemischen Industrie.

Author information

Author notes

  1. Adrian Zander and Sarah Willkomm: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology & Archaea Centre, University of Regensburg, 93053, Regensburg, Germany

    Adrian Zander, Sarah Willkomm, Luisa Egert, Sarah Stöckl & Dina Grohmann

  2. Division of Biosciences, Institute for Structural and Molecular Biology, University College London, WC1E 6BT, London, UK

    Sapir Ofer & Finn Werner

  3. Molecular Biology of Archaea, Institute of Biology II, University of Freiburg, Microbiology, Schaenzlestraße 1, 79104 Freiburg, Germany

    Marleen van Wolferen & Sonja-Verena Albers

  4. Institute of Physical and Theoretical Chemistry – NanoBioSciences, Technische Universität Braunschweig-BRICS, Rebenring 56, 38106 Braunschweig, Germany

    Sabine Buchmeier & Philip Tinnefeld

  5. Department of Chemistry, Center for Integrated Protein Science Munich CIPSM, Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany

    Sabine Schneider

  6. Department Biology I – Plant Development, Biocentre of the LMU Munich, Großhadernerstraße 2-4, 82152 Planegg-Martinstried, Germany

    Andreas Klingl

Authors
  1. Adrian Zander
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah Willkomm
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sapir Ofer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marleen van Wolferen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Luisa Egert
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sabine Buchmeier
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sarah Stöckl
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Philip Tinnefeld
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Sabine Schneider
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Andreas Klingl
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Sonja-Verena Albers
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Finn Werner
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Dina Grohmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.G. conceived the study. A.Z., S.W., M.v.W., L.E., S.St., S.O., A.K., S.B. and D.G. carried out experimental work. A.Z., S.W., M.v.W., S.-V.A., S.Sc., P.T., D.G., A.K. and F.W. performed data analysis. D.G. wrote the manuscript. All authors edited the manuscript.

Corresponding author

Correspondence to Dina Grohmann.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Methods and Supplementary Figures 1–13. (PDF 1676 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zander, A., Willkomm, S., Ofer, S. et al. Guide-independent DNA cleavage by archaeal Argonaute from Methanocaldococcus jannaschii. Nat Microbiol 2, 17034 (2017). https://doi.org/10.1038/nmicrobiol.2017.34

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/nmicrobiol.2017.34

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing