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

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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.

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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

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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.

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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

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

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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

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