Measurements of DNA-loop formation via Cre-mediated recombination

Nucleic Acids Res. 2012 Aug;40(15):7452-64. doi: 10.1093/nar/gks430. Epub 2012 May 15.

Abstract

The Cre-recombination system has become an important tool for genetic manipulation of higher organisms and a model for site-specific DNA-recombination mechanisms employed by the λ-Int superfamily of recombinases. We report a novel quantitative approach for characterizing the probability of DNA-loop formation in solution using time-dependent ensemble Förster resonance energy transfer measurements of intra- and inter-molecular Cre-recombination kinetics. Our method uses an innovative technique for incorporating multiple covalent modifications at specific sites in covalently closed DNA. Because the mechanism of Cre recombinase does not conform to a simple kinetic scheme, we employ numerical methods to extract rate constants for fundamental steps that pertain to Cre-mediated loop closure. Cre recombination does not require accessory proteins, DNA supercoiling or particular metal-ion cofactors and is thus a highly flexible system for quantitatively analyzing DNA-loop formation in vitro and in vivo.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA / chemistry*
  • Fluorescence Resonance Energy Transfer
  • Integrases / chemistry
  • Integrases / metabolism*
  • Kinetics
  • Models, Molecular
  • Nucleic Acid Conformation
  • Recombination, Genetic*

Substances

  • DNA
  • Cre recombinase
  • Integrases