Mutagenesis of PepA suggests a new model for the Xer/cer synaptic complex.

PepA is an aminopeptidase and also functions as a DNA-binding protein in two unrelated systems in Escherichia coli: Xer site-specific recombination and transcriptional regulation of carAB. In these systems, PepA binds to and brings together distant segments of DNA to form interwrapped, nucleosome-like structures. Here we report the selection of PepA mutants that were unable to support efficient Xer recombination. These mutants were defective in DNA-binding and in transcriptional regulation of carAB, but had normal peptidase activity. The mutations define extended patches of basic residues on the surface of the N-terminal domain of PepA that flank a previously proposed DNA-binding groove in the C-terminal domain of PepA. Our results suggest that DNA passes through this C-terminal groove in the PepA hexamer, and is bound by N-terminal DNA-binding determinants at each end of the groove. Based on our data, we propose a new model for the Xer synaptic complex, in which two recombination sites are wrapped around a single hexamer of PepA, bringing the cross-over sites together for strand exchange by the Xer recombinases. In this model, PepA stabilizes negative plectonemic interwrapping between two segments of DNA by passing one segment through the C-terminal groove while the other is held in place in a loop over the groove.