Volume 19, Issue 4, Pages 559-566 (August 2005) The Single-Stranded Genome of Phage CTX Is the Form Used for Integration into the Genome of Vibrio cholerae  Marie-Eve Val, Marie Bouvier, Javier Campos, David Sherratt, François Cornet, Didier Mazel, François-Xavier Barre  Molecular Cell  Volume 19, Issue 4, Pages 559-566 (August 2005) DOI: 10.1016/j.molcel.2005.07.002 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 In Vitro Binding and Cleavage of attP (+) ssDNA by XerC and XerD Xer binding sites are indicated as an upper line; strands that have been shown to be or are expected to be attacked by XerC and/or XerD are shown in red and blue, respectively. Positions of strand cleavage are indicated with triangles; filled triangles indicate experimentally determined cleavage sites, while open triangles correspond to presumed sites. (A) E. coli dif site and its V. cholerae counterparts dif1 and dif2. A dif site consists of two XerC and XerD binding sites separated by a 6 bp central region. (B) Sequence of the double-stranded attP region of CTXϕ replicative form. The two inverted dif-like sequences are connected by a 90 bp DNA sequence indicated as a dotted line. (C) The attP region of the (+) ssDNA of CTXϕ, which is normally packaged in phage particles, can adopt a forked hairpin structure. (D) Scheme of attP1, a synthetic probe mimicking the stem of the attP (+) ssDNA hairpin. (E) Gel retardation analysis using nondenaturing polyacrylamide gel electrophoresis demonstrates that the E. coli XerC and XerD are able to bind to attP1. Two types of complexes are observed, corresponding to either one or two bound Xer recombinases. The nature of each complex is indicated beside the gel. (F) In vitro covalent complex formation between the Xer recombinases and the red or blue strands of attP1 were detected using a 12% PAGE-SDS after heat denaturation of the samples. 5′ labeling of attP1red reveals XerC-covalent binding while 5′ labeling of attP1blue reveals XerD-covalent binding. A scheme of the products is shown beside the gel. Molecular Cell 2005 19, 559-566DOI: (10.1016/j.molcel.2005.07.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Cleavage Point of XerC on attP1 Upon cleavage, XerC forms a covalent complex with the 5′ end of attP1red, which liberates a shorter 3′ end fragment. AttP1red was 3′ labeled and the size of the liberated 3′ end fragment was analyzed on a 12% denaturing polyacrylamide gel. Lane 1: in vitro covalent complexes were generated by XerC-mediated cleavage in the absence of XerD to increase the efficiency of the reaction; lane 2: control in the absence of XerC; lane 3–7: 3′-labeled oligonucleotide ladder encompassing the length of the expected cleaved fragment (open triangle). The filled arrow indicates the cleaved product. Molecular Cell 2005 19, 559-566DOI: (10.1016/j.molcel.2005.07.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Scheme of the Proposed Recombination Reaction between attP (+) ssDNA and V. cholerae dif1 (attB) that Would Result in Integration of the Phage Bases in attP (+) ssDNA that differ from dif1 are shown in bold type. Religation events after strand exchanges are represented with an arc. The reaction stops after a single pair of strand exchange catalyzed by XerC. The branched intermediate is then converted to dsDNA products by cellular processes, such as replication and/or gap repair (bases shown in black). The resulting CTXϕ prophage is flanked by two new sites, attL on the left and attR on the right. Integration of the phage disrupts dif1, but attR, which differs from dif1 by a single base pair (shown by an asterisk), can function as a new chromosome dimer resolution site. Molecular Cell 2005 19, 559-566DOI: (10.1016/j.molcel.2005.07.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 In Vitro Xer Recombination between the ssDNA attP Region of CTXϕ and dif (A) Scheme of the dif probe used in (B) and (C). (B) Xer-mediated recombination between dif and a circular ssDNA containing either the (+) or (−) strand CTXϕ attP region. The 5′ end of difred was radioactively labeled. (C) Only the red strand of dif is exchanged with attP (+) ssDNA. Circular ssDNA containing attP (+) ssDNA was reacted with a dif probe, which was labeled on the 5′ end of difred or difblue. Samples were analyzed by agarose gel electrophoresis after denaturation. (D) Integration of attP1 into a dif-containing plasmid. attP1red was either 5′ or 3′ labeled. Products of recombination were analyzed by agarose gel electrophoresis under their native form (n) or after denaturation (d). Recombination products migrate at a position equivalent to linear ssDNA after denaturation or nicked circle on native conditions. (E) Both recombinases are required for recombination between attP1 and dif, but only the catalytic activity of XerC is necessary. The red strand of attP1 was 5′ end-labeled. attP1 was reacted with a dif-containing plasmid in the presence or absence of XerD and a XerD catalytic mutant (YF). (F) Importance of the secondary structure of the attP (+) ssDNA for its integration into a dif-containing plasmid. Scheme of the different attP probes recombined with a dif-containing plasmid. The red strand of each probe was 5′ labeled. Molecular Cell 2005 19, 559-566DOI: (10.1016/j.molcel.2005.07.002) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 A Strategy for Integration Using Single-Stranded DNA (A) Scheme depicting how CTXϕ (+) ssDNA can be integrated into dif1 using attP (+) ssDNA, and how this site is masked in the dsDNA attL and attR regions of the prophage. Chromosomal DNA is shown in bold, phage DNA in light gray. XerC and XerD are represented as white and gray figures, respectively. Recombination reactions are drawn as if viewed from the C-terminal side of the recombinases to show the C-terminal interactions that control XerCD catalysis. (B) The (+) ssDNA of the attP region of E. coli CUSϕ-1 and Y. pestis CUSϕ-2 can fold into a hairpin, which creates a target for recombination by XerC and XerD similar to CTXϕ attP (+) ssDNA. Molecular Cell 2005 19, 559-566DOI: (10.1016/j.molcel.2005.07.002) Copyright © 2005 Elsevier Inc. Terms and Conditions