Single-molecule microscopy of Cre recombination

Abstract

The Cre recombination system, which has been commonly used as a bioengineering tool for altering eukaryotic genomes in a highly efficient and precise manner, is an archetypical member of a large family of tyrosine site-specific DNA recombinases. Different family members are responsible for a wide range of biological pathways such as regulation of gene expression and DNA replication, plasmid copy number maintenance, conjugative transposition, catenated circle resolution, daughter chromosome segregation, and prokaryotic telomere processing. Despite this wide-ranging biology, it is thought that all family members proceed through a common multistep reaction pathway (Fig. 1A) in which a recombinase tetramer executes a sequential pair of strand exchanges that first generate (Fig. 1A, i–iii) and then resolve (Fig. 1A, iv–vi) a four-way DNA Holliday junction (HJ) intermediate. Recombination is accomplished without high-energy cofactors via two pairs of DNA cleavage and ligation reactions staggered by 6 to 8 bp and mediated by covalent 3′-phosphotyrosine linkages (Fig. 1A, ii and iv). By elegantly pairing two single molecule techniques, Pinkney et al. (1) extract several details from this important model system and provide a framework for investigations into other systems.