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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
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Loop-closure kinetics reveal a stable, right-handed DNA intermediate in Cre recombination.

Massa J Shoura1,2, Stefan M Giovan2, Alexandre A Vetcher2

  • 1Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA.

Nucleic Acids Research
|March 18, 2020
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Summary

Cre recombinase forms a Holliday junction (HJ) intermediate during DNA recombination. This study reveals significant out-of-plane distortions in the HJ structure, challenging previous crystallographic models and highlighting dynamic aspects of this crucial enzymatic process.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • Cre site-specific recombination involves a homotetramer complex with loxP DNA sites.
  • The mechanism proceeds through a Holliday junction (HJ) intermediate.
  • Controversy exists regarding the DNA geometry within the Cre-loxP synapse, with crystallographic data suggesting a planar HJ and topological assays indicating a chiral structure.

Purpose of the Study:

  • To investigate the in-aqueo ensemble of conformations for the DNA looping intermediate in Cre-loxP recombination.
  • To resolve the controversy surrounding the Holliday junction geometry during Cre-mediated recombination.

Main Methods:

  • Utilized kinetics of loop closure assays with closely spaced loxP sites (131-151 bp).
  • Applied statistical-mechanical theory of DNA looping to analyze loop-closure probability (J) as a function of site spacing.
  • Investigated an HJ-isomerization-deficient Cre mutant to understand the dynamic contributions to wild-type complex geometry.

Main Results:

  • Experimental data revealed substantial out-of-plane distortion of the Holliday junction, contradicting static crystallographic models.
  • The observed HJ geometry contrasts sharply with the square-planar structure seen in crystal structures of Cre-loxP and other recombinases.
  • Measurements from a mutant suggested that the wild-type complex's apparent geometry results from temporal averaging of right-handed and achiral structures.

Conclusions:

  • The study provides evidence for dynamic, non-planar Holliday junction structures in Cre-loxP recombination, reconciling conflicting experimental observations.
  • This dynamic analysis offers a more comprehensive understanding of large nucleoprotein structures and their mechanisms beyond static crystal structures.