Change in topological linking number during Xer recombination at the plasmid pSC101 psi site

Abstract

The XerCD recombinase functions with accessory proteins PepA, ArcA and ArgR at plasmid recombination sites such as psi and cer to ensure stable monomeric plasmid inheritance. Xer recombination acts only on directly repeated psi sites, and recombination produces a specific catenane of two product circles interlinked exactly four times. Here we measure the precise change in topological linkage ({Delta}Lk) that occurs during Xer recombination at psi. We use a DNA substrate with close-spaced psi sites that recombines to produce one circle of 398 bp and another of 3039 bp and demonstrate that the small circle is exclusively the -1 topoisomer. Using a purified topoisomer of the substrate, we show that Xer recombination proceeds with a linkage change ({Delta}Lk) of +4. Similar experiments using a substrate with equally spaced psi sites agreed with this result. The measured linkage change is consistent with a reaction mechanism for tyrosine recombinases in which the sites align antiparallel prior to recombination and recombine via a Holliday junction intermediate. Four negative supercoils are converted to catenation nodes by strand exchange, providing an energetic driving force for the reaction. We compare this result to the mechanisms of serine recombinases and the recently discovered bridge RNA-guided recombinases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/706985v2_ufig1.gif" ALT="Figure 1000"> View larger version (29K): org.highwire.dtl.DTLVardef@3ac1c5org.highwire.dtl.DTLVardef@1876da7org.highwire.dtl.DTLVardef@301ff0org.highwire.dtl.DTLVardef@180e054_HPS_FORMAT_FIGEXP M_FIG C_FIG