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. 2012 Aug 3;31(18):3757–3767. doi: 10.1038/emboj.2012.219

Figure 4.

Figure 4

Endo III stabilizes the pseudo-HJ integration intermediate of CTXϕ. (A) Scheme of the life cycle of CTXϕ. 1: adsorption to the Toxin-Coregulated Pilus (TCP); 2: conversion of the circular (+) ssDNA phage genome into its dsDNA replicative form; 3: production of (+) ssDNA by RCR; 4: Integration of the ssDNA by Xer recombination; 5: production of new extrachromosomal copies of the phage genome by a process analogous to RCR between two tandem copies of the prophage; 6: encapsidation of the (+) ssDNA and production of new virions. (B) Efficiency of integration of a non-replicative vector harbouring RS1 attP. Integration was monitored directly after conjugation. Mean and standard deviation of at least three independent experiments. (C) Scheme of the three reversible steps of the recombination reaction leading to the formation of the attP/dif pseudo-HJ. Legend is as in Figure 1. (i) XerC-mediated cleavage; (ii) exchange of the liberated 5′-hydroxyl extremities; and (iii) self-re-ligation or re-ligation to the partner recombining strand. (D) In-vitro equilibrium reached after 4 h of incubation of a short radioactively labelled dif1 substrate and a longer cold attP(+) substrate with the Xer recombinases. The shorter migration product reveals cleavage of the recombining dif1 strand. The longer migration product reveals formation of the attP/dif pseudo-HJ formation. Unlabelled oligonucleotides cannot be directly visualized and do not interfere with the migration of the labelled fragments in the denaturing gel. Schemes of substrate and products are drawn on the left of the gels. A star indicates the position of the radioactive label. Unlabelled oligonucleotides are represented as dashed lines. (E) Cleaved and resolved products obtained after 1 h of incubation of labelled attP(+)/dif pseudo-HJs with the Xer recombinases. Samples were treated with proteinase K to avoid any interference of the covalent 3′-phosphotyrosyl bonds between XerC and the cleaved fragments in the oligonucleotide migration. First and second indicate the order of addition of Endo III and of the Xer recombinases.