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.
Reijns
Martin
M
Institute of Biomedical and Life Sciences, Division of Molecular Genetics, University of Glasgow, Anderson College, 56 Dumbarton Road, Glasgow G11 6NU, Scotland, UK.
Lu
Yangjie
Y
Leach
Stephen
S
Colloms
Sean D
SD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019426:Integrases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011995:Recombination, Genetic; D012097:Repressor Proteins / Q000378:metabolism; D014158:Transcription, Genetic
C490177:ArgR protein, E coli; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C496211:PepA protein, E coli; D012097:Repressor Proteins; C473360:XerC protein, E coli; D019426:Integrases; C473361:XerD protein, E coli