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

Measurements of DNA-loop formation via Cre-mediated recombination.

Author information

1
Department of Molecular and Cell Biology, University of Texas at Dallas, 800 West Campbell Road, Richardson, TX 75080, USA.

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.

PMID:
22589415
PMCID:
PMC3424569
DOI:
10.1093/nar/gks430
[Indexed for MEDLINE]
Free PMC Article

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