Enzymatic formation of biparental figure-eight molecules from plasmid DNA and their resolution in E. coli

Abstract

A key intermediate in general genetic recombination is a structure in which two double-stranded DNA molecules are covalently linked by a single-strand crossover characteristic of a Holliday junction. When the DNA molecules are circular, the recombinant structures take the form of a figure eight. We have used purified E. coli enzymes to construct biparental figure-eight DNA molecules in vitro from the DNA of two partially homologous plasmids. When purified figure-eight structures are transfected into recA- E. coli cells, they are resolved to produce monomeric or dimeric plasmid progeny, apparently by the cutting and joining of the Holliday crossover. The maturation of figure-eight molecules in bacteria is characterized by the formation and recovery of both parental and recombinant types, cross-over at a frequency of up to 50% and the capability for mismatch repair at regions of hybrid DNA. In these three regards, the products of figure-eight maturation resemble recombinant chromosomes formed at meiosis. These observations show that biparental figure eights behave as recombination intermediates that can be resolved into mature recombinants without need for a functional recA+ gene product.