Skip to main content
SpringerLink
Log in

Molecular Genetic Methods to Study DNA Replication Protein Function in Haloferax volcanii, A Model Archaeal Organism

  • Protocol
DNA Replication

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

Abstract

Successful high-fidelity chromosomal DNA replication is fundamental to all forms of cellular life and requires the complex interplay of a variety of essential and nonessential protein factors in a spatially and temporally coordinated manner. Much of what is known about the enzymes and mechanisms of chromosome replication has come from analysis of simple microbial model systems, such as yeast and archaea. Archaea possess a highly simplified eukaryotic-like replication apparatus, making them an excellent model for gaining novel insights into conserved aspects of protein function at the heart of the replisome. Amongst the thermophilic archaea, a number of species have proved useful for biochemical analysis of protein function, but few of these organisms are suited to genetic analysis. One archaeal organism that is genetically tractable is the mesophilic euryarchaeon Haloferax volcanii, a halophile that grows aerobically in high salt medium at an optimum temperature of 40–45 °C and with a doubling time of 2–3 h. The Hfx. volcanii genome has been sequenced and a range of methods have been developed to allow reverse genetic analysis of protein function in vivo, including techniques for gene replacement and gene deletion, transcriptional regulation, point mutation and gene tagging. Here we briefly summarize current knowledge of the chromosomal DNA replication machinery in the haloarchaea before describing in detail the molecular methods available to probe protein structure and function within the Hfx. volcanii replication apparatus.

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 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

Institutional subscriptions

References

  1. Bicknell LS, Bongers EM, Leitch A, Brown S, Schoots J, Harley ME, Aftimos S, Al-Aama JY, Bober M, Brown PA, van Bokhoven H, Dean J, Edrees AY, Feingold M, Fryer A, Hoefsloot LH, Kau N, Knoers NV, Mackenzie J, Opitz JM, Sarda P, Ross A, Temple IK, Toutain A, Wise CA, Wright M, Jackson AP (2011) Mutations in the pre-replication complex cause Meier-Gorlin syndrome. Nat Genet 43:356–359

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Bicknell LS, Walker S, Klingseisen A, Stiff T, Leitch A, Kerzendorfer C, Martin CA, Yeyati P, Al Sanna N, Bober M, Johnson D, Wise C, Jackson AP, O’Driscoll M, Jeggo PA (2011) Mutations in ORC1, encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. Nat Genet 43:350–355

    Article  CAS  PubMed  Google Scholar 

  3. Guernsey DL, Matsuoka M, Jiang H, Evans S, Macgillivray C, Nightingale M, Perry S, Ferguson M, LeBlanc M, Paquette J, Patry L, Rideout AL, Thomas A, Orr A, McMaster CR, Michaud JL, Deal C, Langlois S, Superneau DW, Parkash S, Ludman M, Skidmore DL, Samuels ME (2011) Mutations in origin recognition complex gene ORC4 cause Meier-Gorlin syndrome. Nat Genet 43:360–364

    Article  CAS  PubMed  Google Scholar 

  4. Shima N, Alcaraz A, Liachko I, Buske TR, Andrews CA, Munroe RJ, Hartford SA, Tye BK, Schimenti JC (2007) A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice. Nat Genet 39:93–98

    Article  CAS  PubMed  Google Scholar 

  5. Barry ER, Bell SD (2006) DNA replication in the archaea. Microbiol Mol Biol Rev 70:876–887

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Liu J, Smith CL, DeRyckere D, DeAngelis K, Martin GS, Berger JM (2000) Structure and function of Cdc6/Cdc18: implications for origin recognition and checkpoint control. Mol Cell 6:637–648

    Article  CAS  PubMed  Google Scholar 

  7. Brewster AS, Wang G, Yu X, Greenleaf WB, Carazo JM, Tjajadi M, Klein MG, Chen XS (2008) Crystal structure of a near-full-length archaeal MCM: functional insights for an AAA+ hexameric helicase. Proc Natl Acad Sci 105:20191–20196

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Lao-Sirieix SH, Nookala RK, Roversi P, Bell SD, Pellegrini L (2005) Structure of the heterodimeric core primase. Nat Struct Mol Biol 12:1137–1144

    Article  CAS  PubMed  Google Scholar 

  9. Zhao Y, Jeruzalmi D, Moarefi I, Leighton L, Lasken R, Kuriyan J (1999) Crystal structure of an archaebacterial DNA polymerase. Structure 7:1189–1199

    Article  CAS  PubMed  Google Scholar 

  10. Hopfner KP, Eichinger A, Engh RA, Laue F, Ankenbauer W, Huber R, Angerer B (1999) Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius. Proc Natl Acad Sci 96:3600–3605

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Matsumiya S, Ishino Y, Morikawa K (2001) Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus. Protein Sci 10:17–23

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Hosfield DJ, Mol CD, Shen B, Tainer JA (1998) Structure of the DNA repair and replication endonuclease and exonuclease FEN-1: coupling DNA and PCNA binding to FEN-1 activity. Cell 95:135–146

    Article  CAS  PubMed  Google Scholar 

  13. Nishida H, Kiyonari S, Ishino Y, Morikawa K (2006) The closed structure of an archaeal DNA ligase from Pyrococcus furiosus. J Mol Biol 360:956–967

    Google Scholar 

  14. Farkas JA, Picking JW, Santangelo TJ (2013) Genetic techniques for the archaea. Annu Rev Genet 47:539–561

    Article  CAS  PubMed  Google Scholar 

  15. Mullakhanbhai MF, Larsen H (1975) Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Arch Microbiol 104(3):207–214

    Google Scholar 

  16. Hartman AL, Norais C, Badger JH, Delmas S, Haldenby S, Madupu R, Robinson J, Khouri H, Ren Q, Lowe TM, Maupin-Furlow J, Pohlschroder M, Daniels C, Pfeiffer F, Allers T, Eisen JA (2010) The complete genome sequence of Haloferax volcanii DS2, a model archaeon. PLoS One 5:e9605

    Article  PubMed Central  PubMed  Google Scholar 

  17. Hawkins M, Malla S, Blythe MJ, Nieduszynski CA, Allers T (2013) Accelerated growth in the absence of DNA replication origins. Nature 503:544–547

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Norais C, Hawkins M, Hartman AL, Eisen JA, Myllykallio H, Allers T (2007) Genetic and physical mapping of DNA replication origins in Haloferax volcanii. PLoS Genet 3:e77

    Article  PubMed Central  PubMed  Google Scholar 

  19. Kristensen TP, Maria Cherian R, Gray FC, MacNeill SA (2014) The haloarchaeal MCM proteins: bioinformatic analysis and targeted mutagenesis of the β7-β8 and β9-β10 hairpin loops and conserved zinc binding domain cysteines. Front Microbiol 5:123

    Article  PubMed Central  PubMed  Google Scholar 

  20. Skowyra A, MacNeill SA (2012) Identification of essential and non-essential single-stranded DNA-binding proteins in a model archaeal organism. Nucleic Acids Res 40:1077–1090

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Stroud A, Liddell S, Allers T (2012) Genetic and biochemical identification of a novel single-stranded DNA-binding complex in Haloferax volcanii. Front Microbiol 3:224

    Google Scholar 

  22. Morgunova E, Gray FC, MacNeill SA, Ladenstein R (2009) Structural insights into the adaptation of proliferating cell nuclear antigen (PCNA) from Haloferax volcanii to a high-salt environment. Acta Crystallogr 65:1081–1088

    CAS  Google Scholar 

  23. Winter JA, Christofi P, Morroll S, Bunting KA (2009) The crystal structure of Haloferax volcanii proliferating cell nuclear antigen reveals unique surface charge characteristics due to halophilic adaptation. BMC Struct Biol 9:55

    Article  PubMed Central  PubMed  Google Scholar 

  24. Poidevin L, MacNeill SA (2006) Biochemical characterisation of LigN, an NAD + -dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations. BMC Mol Biol 7:44

    Article  PubMed Central  PubMed  Google Scholar 

  25. Zhao A, Gray FC, MacNeill SA (2006) ATP- and NAD+-dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii. Mol Microbiol 59:743–752

    Article  CAS  PubMed  Google Scholar 

  26. Allers T, Ngo HP, Mevarech M, Lloyd RG (2004) Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Appl Environ Microbiol 70:943–953

    Google Scholar 

  27. Wendoloski D, Ferrer C, Dyall-Smith ML (2001) A new simvastatin (mevinolin)-resistance marker from Haloarcula hispanica and a new Haloferax volcanii strain cured of plasmid pHV2. Microbiology 147:959–964

    CAS  PubMed  Google Scholar 

  28. Pfeiffer F, Broicher A, Gillich T, Klee K, Mejia J, Rampp M, Oesterhelt D (2008) Genome information management and integrated data analysis with HaloLex. Arch Microbiol 190:281–299

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Dyall-Smith ML. The Halohandbook: protocols for haloarchaeal genetics. http://www.haloarchaea.com/resources/halohandbook/

  30. Bitan-Banin G, Ortenberg R, Mevarech M (2003) Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene. J Bacteriol 185:772–778

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  31. Scherer S, Davis RW (1979) Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci 76:4951–4955

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Large A, Stamme C, Lange C, Duan Z, Allers T, Soppa J, Lund PA (2007) Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene. Mol Microbiol 66:1092–1106

    Article  CAS  PubMed  Google Scholar 

  33. Mevarech M, Werczberger R (1985) Genetic transfer in Halobacterium volcanii. J Bacteriol 162:461–462

    PubMed Central  CAS  PubMed  Google Scholar 

  34. Rosenshine I, Tchelet R, Mevarech M (1989) The mechanism of DNA transfer in the mating system of an archaebacterium. Science 245:1387–1389

    Article  CAS  PubMed  Google Scholar 

  35. Allers T, Barak S, Liddell S, Wardell K, Mevarech M (2010) Improved strains and plasmid vectors for conditional overexpression of His-tagged proteins in Haloferax volcanii. Appl Environ Microbiol 76:1759–1769

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Breuert S, Allers T, Spohn G, Soppa J (2006) Regulated polyploidy in halophilic archaea. PLoS One 1:e92

    Article  PubMed Central  PubMed  Google Scholar 

  37. Le Breton M, Henneke G, Norais C, Flament D, Myllykallio H, Querellou J, Raffin JP (2007) The heterodimeric primase from the euryarchaeon Pyrococcus abyssi: a multifunctional enzyme for initiation and repair? J Mol Biol 374:1172–1185

    Article  PubMed  Google Scholar 

  38. Meslet-Cladiere L, Norais C, Kuhn J, Briffotaux J, Sloostra JW, Ferrari E, Hubscher U, Flament D, Myllykallio H (2007) A novel proteomic approach identifies new interaction partners for proliferating cell nuclear antigen. J Mol Biol 372:1137–1148

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to our colleagues past and present in Copenhagen, St Andrews and elsewhere for their contributions to developing the techniques described in this chapter. Archaeal research in the MacNeill lab has been funded by SULSA (Scottish Universities Life Science Alliance), the US Air Force Office of Scientific Research (award number FA9550-10-1-0421), and Forskningsrådet for Natur og Univers (FNU sagsnr. 272-05-0446).

Author information

Authors and Affiliations

  1. Sorbonne Paris Cité, Faculté de Médecine Paris Descartes, Université Paris Descartes, Paris, France

    Xavier Giroux

  2. Biomedical Sciences Research Complex, School of Biology, University of St Andrews, North Haugh, St Andrews, Fife, KY16 9ST, UK

    Stuart A. MacNeill

Authors
  1. Xavier Giroux
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stuart A. MacNeill
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stuart A. MacNeill .

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

Giroux, X., MacNeill, S.A. (2015). Molecular Genetic Methods to Study DNA Replication Protein Function in Haloferax volcanii, A Model Archaeal Organism. 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_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2596-4_13

  • 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