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

Abstract

SummaryPepA 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 crossover 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.

Self‐control in DNA site‐specific recombination mediated by the tyrosine recombinase TnpI

“…In the recombination complex of other resolvases of the serine recombinase family, one recombinase dimer is replaced by a non-specific DNA bending protein like HU or Hbsu, but the core architecture of the synapse is the same as that proposed for res (Rowland et al, 2002;Canosa et al, 2003). A structurally distinct, but topologically equivalent organization was also proposed for the synaptic complex formed around the accessory proteins at the Xer recombination sites cer and psi (Alén et al, 1997;Colloms et al, 1997;Reijns et al, 2005). Recombination at psi exclusively yields four-noded catenanes .…”

“…A similar mechanism was latterly proposed for the resolution of plasmid multimers by the tyrosine recombinase XerCD . In this case, formation of the productive synapse is controlled by wrapping the recombination sites around a complex formed by the host proteins PepA and ArgR, or PepA and ArcA (Alèn et al ., 1997;Gourlay and Colloms, 2004;Reijns et al ., 2005).…”

Mutational Analysis of Intervening Sequences Connecting the Binding Sites for Integration Host Factor, PepA, PurR, and RNA Polymerase in the Control Region of the Escherichia coli carAB Operon, Encoding Carbamoylphosphate Synthase

“…The DNAbinding mode of PepA is not well characterized and atypical in the sense that the protein bears no classical DNA-binding motif (24). Separation-of-function pepA mutant analyses indicated the importance of the amino-terminal domain of the enzyme for DNA binding (7,22). The same analyses revealed the existence of pepA mutants deficient for the regulatory response but not affected in the DNA-binding capacity and proficient for the resolution reaction.…”

“…PepA binds to the carAB control region and also to its own control region (6) and to the ColE1 cer site (1,22), where it imposes in conjunction with ArgR the directionality of the site-specific resolution reaction of ColE1 multimers (15,23). The aminopeptidase activity of the enzyme is neither required for transcriptional regulation nor for the resolution reaction (6,20).…”

Differences in Resolution of mwr -Containing Plasmid Dimers Mediated by the Klebsiella pneumoniae and Escherichia coli XerC Recombinases: Potential Implications in Dissemination of Antibiotic Resistance Genes

“…Recombination at plasmid sites such as psi (pSC101), cer (ColE1), or mwr (pJHCMW1) requires a core site related to dif plus an adjacent DNA stretch of about 180 bp known as accessory sequences (20,46,49). The proteins PepA and ArgR (in the case of cer and mwr) or PepA and ArcA (in the case of psi) bind the accessory sequences and induce the formation of a synaptic complex required for recombination and for ensuring that the reaction is exclusively intramolecular (1,12,17,18,27,35). Xer recombination at plasmid sites starts with the exchange of one pair of strands catalyzed by XerC, resulting in formation of a Holliday junction.…”