Skip to main content
SpringerLink
Log in

Insights into the preferential order of strand exchange in the Cre/loxP recombinase system: impact of the DNA spacer flanking sequence and flexibility

  • Published:
Journal of Computer-Aided Molecular Design Aims and scope Submit manuscript

Abstract

The Cre/loxP system is widely used as a genetic tool to manipulate DNA. Cre recombinase catalyzes site-specific recombination between 34 bp loxP sites. Each loxP site is recognized by two Cre molecules assuming a cleaving (CreC) and non-cleaving (CreNC) activity. Despite the symmetry in the sequences of the arms of loxP, available biochemical data show strong evidence that the recombination reaction is asymmetric with a preferred strand exchange order. The asymmetry comes from the spacer separating the two sets of palindromic arms of the loxP sequence. However, it remains to be understood how this preferential order is established. We apply computational structure-based methods and perform a thorough detailed analysis of available structural and biochemical information on the Cre/loxP system in order to investigate such asymmetry in the recombination, and we propose a rationale to explain the determinants favoring the strand exchange order. We show that the structural properties of the DNA flanking sequence of the spacer guide the recombination, and we establish the role of residues R118, R121 and K122 from CreC, which contact the spacer region and by clamping the DNA inhibit the cleavage on the second arm of loxP. Our studies give an atomistic insight on the synapsis state of the recombination process in the Cre/loxP system and highlight the importance of the flexibility and other intrinsic properties of the flanking regions of the DNA spacer to establish a preferential strand exchange order.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

CreC :

Cleaving Cre recombinase

CreNC :

Non-cleaving Cre recombinase

bp:

DNA base pair

Sp:

DNA spacer

HJ:

DNA Holliday junction

BS:

Bottom DNA strand

TS:

Top DNA strand

NpN:

Dinucleotide step

MD:

Molecular dynamics

PDB:

Brookhaven Protein data bank

RMSD:

Root-mean-square deviation of atomic positions

RMSDbck :

Root-mean-square deviation of backbone atomic positions

Hb:

Hydrogen bond

ONIOM:

Gaussian’s Our own N-layered Integrated molecular Orbital and molecular Mechanics

AMBER:

Assisted Model Building with Energy Refinement

TRX scale:

Twist, Roll, and X-disp (base pair displacement) scale

QM:

Quantum mechanics

MM:

Molecular Mechanics

QM/MM:

Hybrid quantum mechanics/molecular mechanics

QM/MD:

Hybrid quantum mechanics/molecular dynamics

mG:

DNA minor groove

References

  1. Grindley ND, Whiteson KL, Rice PA (2006) Mechanisms of site-specific recombination. Annu Rev Biochem 75:567–605

    Article  CAS  Google Scholar 

  2. Hoess R, Wierzbicki A, Abremski K (1987) Isolation and characterization of intermediates in site-specific recombination. Proc Natl Acad Sci USA 84(19):6840–6844

    Article  CAS  Google Scholar 

  3. Ghosh K, Lau CK, Gupta K, Van Duyne GD (2005) Preferential synapsis of loxP sites drives ordered strand exchange in Cre–loxP site-specific recombination. Nat Chem Biol 1(5):275–282

    Article  CAS  Google Scholar 

  4. Lee L, Chu LC, Sadowski PD (2003) Cre induces an asymmetric DNA bend in its target loxP site. J Biol Chem 278(25):23118–23129

    Article  CAS  Google Scholar 

  5. Lee L, Sadowski PD (2001) Directional resolution of synthetic holliday structures by the Cre recombinase. J Biol Chem 276(33):31092–31098

    Article  CAS  Google Scholar 

  6. Lee L, Sadowski PD (2003) Sequence of the loxP site determines the order of strand exchange by the Cre recombinase. J Mol Biol 326(2):397–412

    Article  CAS  Google Scholar 

  7. Lee G, Saito I (1998) Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216(1):55–65

    Article  CAS  Google Scholar 

  8. Ghosh K, Guo F, Van Duyne GD (2007) Synapsis of loxP sites by Cre recombinase. J Biol Chem 282(33):24004–24016

    Article  CAS  Google Scholar 

  9. Ennifar E, Meyer JE, Buchholz F, Stewart AF, Suck D (2003) Crystal structure of a wild-type Cre recombinase–loxP synapse reveals a novel spacer conformation suggesting an alternative mechanism for DNA cleavage activation. Nucl Acid Res 31(18):5449–5460

    Article  CAS  Google Scholar 

  10. Gelato KA, Martin SS, Baldwin EP (2005) Reversed DNA strand cleavage specificity in initiation of Cre–loxP recombination induced by the His289Ala active-site substitution. J Mol Biol 354(2):233–245

    Article  CAS  Google Scholar 

  11. Martin SS, Pulido E, Chu VC, Lechner TS, Baldwin EP (2002) The order of strand exchanges in Cre–loxP recombination and its basis suggested by the crystal structure of a Cre–loxP Holliday junction complex. J Mol Biol 319(1):107–127

    Article  CAS  Google Scholar 

  12. Van Duyne GD (2001) A structural view of Cre–loxp site-specific recombination. Annu Rev Biophys Biomol Struct 30:87–104

    Article  Google Scholar 

  13. Guo F, Gopaul DN, van Duyne GD (1997) Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature 389(6646):40–46

    Article  CAS  Google Scholar 

  14. Guo F, Gopaul DN, Van Duyne GD (1999) Asymmetric DNA bending in the Cre–loxP site-specific recombination synapse. Proc Natl Acad Sci USA 96(13):7143–7148

    Article  CAS  Google Scholar 

  15. Pinkney JN, Zawadzki P, Mazuryk J, Arciszewska LK, Sherratt DJ, Kapanidis AN (2012) Capturing reaction paths and intermediates in Cre–loxP recombination using single-molecule fluorescence. Proc Natl Acad Sci USA 109(51):20871–20876

    Article  CAS  Google Scholar 

  16. Heddi B, Foloppe N, Oguey C, Hartmann B (2008) Importance of accurate DNA structures in solution: the Jun–Fos model. J Mol Biol 382(4):956–970

    Article  CAS  Google Scholar 

  17. Heddi B, Foloppe N, Bouchemal N, Hantz E, Hartmann B (2006) Quantification of DNA BI/BII backbone states in solution. Implications for DNA overall structure and recognition. J Am Chem Soc 128(28):9170–9177

    Article  CAS  Google Scholar 

  18. Heddi B, Foloppe N, Hantz E, Hartmann B (2007) The DNA structure responds differently to physiological concentrations of K+ or Na+. J Mol Biol 368(5):1403–1411

    Article  CAS  Google Scholar 

  19. Winger RH, Liedl KR, Pichler A, Hallbrucker A, Mayer E (1999) Helix morphology changes in B-DNA induced by spontaneous B(I) ⟺ B(II) substrate interconversion. J Biomol Struct Dyn 17(2):223–235

    Article  CAS  Google Scholar 

  20. Djuranovic D, Hartmann B (2004) DNA fine structure and dynamics in crystals and in solution: the impact of BI/BII backbone conformations. Biopolymers 73(3):356–368

    Article  CAS  Google Scholar 

  21. Svozil D, Kalina J, Omelka M, Schneider B (2008) DNA conformations and their sequence preferences. Nucl Acid Res 36(11):3690–3706

    Article  CAS  Google Scholar 

  22. Heddi B, Abi-Ghanem J, Lavigne M, Hartmann B (2010) Sequence-dependent DNA flexibility mediates DNase I cleavage. J Mol Biol 395(1):123–133

    Article  CAS  Google Scholar 

  23. Lee L, Sadowski PD (2003) Identification of Cre residues involved in synapsis, isomerization, and catalysis. J Biol Chem 278(38):36905–36915

    Article  CAS  Google Scholar 

  24. Rohs R, West SM, Sosinsky A, Liu P, Mann RS, Honig B (2009) The role of DNA shape in protein-DNA recognition. Nature 461(7268):1248–1253

    Article  CAS  Google Scholar 

  25. Hizver J, Rozenberg H, Frolow F, Rabinovich D, Shakked Z (2001) DNA bending by an adenine–thymine tract and its role in gene regulation. Proc Natl Acad Sci USA 98(15):8490–8495

    Article  CAS  Google Scholar 

  26. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79:926–935

    Article  CAS  Google Scholar 

  27. Mahoney M, Jorgensen W (2000) A five-site model for liquid water and the reproduction of the density anomaly by rigid, nonpolarizable potential functions. J Chem Phys 112(20):8910–8922

    Article  CAS  Google Scholar 

  28. Berendsen HJC, Postma JPM, van Gunsteren WF, Dinola A, Haak JR (1984) Molecular dynamics with coupling to an external bath. J Chem Phys 81(8):3684–3690

    Article  CAS  Google Scholar 

  29. van Gunsteren WF, Berendsen HJC (1977) Algorithms for macromolecular dynamics and constraint dynamics. Mol Phys 34(5):1311–1327

    Article  Google Scholar 

  30. Case DA, Darden TA, Cheatham TE III, Simmerling CL, Wang J, Duke RE, Luo R, Crowley M, Walker RC, Zhang W, Merz KM, Wang B, Hayik S, Roitberg A, Seabra G, Kolossváry I, Wong KF, Paesani F, Vanicek J, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Mathews DH, Seetin MG, Sagui C, Babin V, Kollman PA (2008) AMBER 10. University of California, San Francisco

    Google Scholar 

  31. Perez A, Marchan I, Svozil D, Sponer J, Cheatham TE 3rd, Laughton CA, Orozco M (2007) Refinement of the AMBER force field for nucleic acids: improving the description of alpha/gamma conformers. Biophys J 92(11):3817–3829. doi:10.1529/biophysj.106.097782

    Article  CAS  Google Scholar 

  32. Zgarbova M, Luque FJ, Sponer J, Cheatham TE 3rd, Otyepka M, Jurecka P (2013) Toward improved description of DNA backbone: revisiting epsilon and zeta torsion force field parameters. J Chem Theory Comput 9(5):2339–2354. doi:10.1021/ct400154j

    Article  CAS  Google Scholar 

  33. Heddi B, Oguey C, Lavelle C, Foloppe N, Hartmann B (2010) Intrinsic flexibility of B-DNA: the experimental TRX scale. Nucl Acid Res 38(3):1034–1047

    Article  CAS  Google Scholar 

  34. Bansal M, Bhattacharyya D, Ravi B (1995) NUPARM and NUCGEN: software for analysis and generation of sequence dependent nucleic acid structures. Comput Appl Biosci 11(3):281–287

    CAS  Google Scholar 

  35. Abi-Ghanem J, Heddi B, Foloppe N, Hartmann B (2010) DNA structures from phosphate chemical shifts. Nucl Acid Res 38(3):e18

    Article  Google Scholar 

  36. McDonald IK, Thornton JM (1994) Satisfying hydrogen bonding potential in proteins. J Mol Biol 238(5):777–793

    Article  CAS  Google Scholar 

  37. Luscombe NM, Laskowski RA, Thornton JM (2001) Amino acid-base interactions: a three-dimensional analysis of protein–DNA interactions at an atomic level. Nucl Acid Res 29(13):2860–2874

    Article  CAS  Google Scholar 

  38. Lavery R, Moakher M, Maddocks JH, Petkeviciute D, Zakrzewska K (2009) Conformational analysis of nucleic acids revisited: curves+. Nucl Acid Res 37(17):5917–5929

    Article  CAS  Google Scholar 

  39. Team RC, (2012) R: a language and environment for statistical computing

  40. Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38 27–38

    Article  CAS  Google Scholar 

  41. Dapprich S, Komáromi I, Byun KS, Morokuma K, Frisch MJ (1999) A new ONIOM implementation in Gaussian98. Part I. The calculation of energies, gradients, vibrational frequencies and electric field derivatives. J Mol Struct 462:1–21

    Article  Google Scholar 

  42. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V, Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Vreven T, Montgomery JA, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC, Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL, Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas O, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ (2009) Gaussian 09,Revision B.01. Gaussian, Inc., Wallingford

    Google Scholar 

  43. Becke AD (1993) A new mixing of Hartree–Fock and local density–functional theories. J Chem Phys 98(2):1372–1377. doi:10.1063/1.464304

    Article  CAS  Google Scholar 

  44. Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, Spellmeyer DC, Fox T, Caldwell JW, Kollman PA (1995) A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J Am Chem Soc 117(19):5179–5197

    Article  CAS  Google Scholar 

  45. Tao P, Schlegel HB (2010) A toolkit to assist ONIOM calculations. J Comput Chem 31(12):2363–2369

    CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Ralf Gey for technical support and the ZIH TU Dresden for high-performance computational resources and assistance. We are grateful to Prof. Frank Buchholz, Dr. Maciej Paszkowski-Rogacz, Dr. Janet Chusainow and Dr. Madina Karimova for valuable discussions.

Author information

Author notes

  1. Josephine Abi-Ghanem

    Present address: CNRS, UMS 3033, IECB, 33600, Pessac, France

Authors and Affiliations

  1. Structural Bioinformatics, Biotec TU Dresden, Tatzberg 47-51, 01037, Dresden, Germany

    Josephine Abi-Ghanem, Sergey A. Samsonov & M. Teresa Pisabarro

Authors
  1. Josephine Abi-Ghanem
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sergey A. Samsonov
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. Teresa Pisabarro
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Josephine Abi-Ghanem or M. Teresa Pisabarro.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Abi-Ghanem, J., Samsonov, S.A. & Pisabarro, M.T. Insights into the preferential order of strand exchange in the Cre/loxP recombinase system: impact of the DNA spacer flanking sequence and flexibility. J Comput Aided Mol Des 29, 271–282 (2015). https://doi.org/10.1007/s10822-014-9825-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10822-014-9825-0

Keywords

Search

Navigation