High efficiency of recombination of two other Rad52 recombinases.

<p>A: Principle of the assay: two 800 bp homologous sequences (<i>oxa</i> genes, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004181#pgen.1004181-Martinsohn1" target="_blank">[19]</a>), inversely repeated, represented by the red and yellow rectangles, are inserted so as to flank the P<i>_L</i> promoter in the λ genome. Inverted sequences are either 100% or 78% identical. When homologous recombination occurs between the repeated sequences, the P<i>_L</i> promoter is inverted, which leads to a phenotypic switch, because the <i>red</i> and <i>gam</i> genes are no longer expressed (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004181#s4" target="_blank">Materials and Methods</a>). B: Recombination frequencies scored with three different pairs of phage recombinase/exonuclease, and compared to RecA pathway. The <i>recET</i> (from Rac prophage) and <i>erf/exo</i> (from phage PA73) genes were substituted in place of the λ <i>red</i> genes. Values shown for <i>redαβ</i> and <i>recA</i> pathways are those reported in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004181#pgen.1004181-Martinsohn1" target="_blank">[19]</a>. Mean ± standard deviation of at least 3 independent recombination assays are indicated.</p

Data collected on mosaics, by categories of genome pairs compared.

<p>*T, temperate, D, defective, V, virulent.</p

Formation of Φ80 hybrids also depend on phage recombinase.

<p>A: map of the 300 bp region of 96% identity between the Φ80 region near attP site and <i>E. coli</i> gene <i>yecD</i>. B: Map of the 980 bp homology region between Dlp12 and Φ80, spanning through the essential terminase genes <i>A</i> and <i>nu1</i>. C: Highly divergent 500 bp homology region between the non-essential nin region of Φ80 and Dlp12. D: Frequency of recombinants for each homology region, as a function of Redβ and RecA (genotypes are indicated below the bars). Mean ± standard deviation of at least 3 independent recombination assays are indicated.</p

Rationale for the bio-informatics detection of mosaics.

<p>Upper panel: two ancestral phages A and B recombine across two regions of partial identity (light grey squares). As a consequence, the new piece of DNA in the recombined phage C, when compared to its parent B providing the mosaic, exhibits a 100% identity region (dark grey square), flanked by the two partially identical sequences (light grey), above the background level <i>b</i> of low identity shared by B and C. Upon alignment of genome C with B around the mosaic (lower panel), if the regions flanking the mosaic have a percentage of identity above the background level of identity (≥<i>b</i>+10%), they will be counted as traces of homologous recombination (HR trace).</p

Position of recombination events in homology regions between λ and defective prophages.

<p>A: Positions of recombination events between Rac and λ for 12 sequenced clones. Mismatches in similarity regions between rac and λ are represented by vertical black bars. Intervals between two mismatches in which sequencing revealed that recombination occurred are colored in green, intensity representing the number of recombination events (see legend). B: Positions of recombination events between Dlp12 and λ. Nine and 19 clones were sequenced on the left and right sides of <i>cat</i> gene, respectively. Half of the recombination events scored occurred at direct proximity of the <i>cos</i> site.</p

Heat map of mosaic characteristics in temperate and defective phages.

<p>Density of mosaics found among pairs of phages (lower part of the diagonal, purple to green colours), and average number of traces of recombination detected at the boundary of mosaics (higher part, yellow to red colours). The density is the number of mosaics per 10 kb of phage genome. Names of λ-like phages are in red.</p

Recombinants between λ and defective prophages are formed during lytic cycle.

<p>A, B and C: Maps of the three regions of similarity between λ and MG1655 used in this study. Antibiotic resistance genes (white arrows) were inserted in the defective prophages, in the middle of the identity regions. <i>KanR</i> stands for the gene conferring resistance to kanamycine, the <i>cat</i> gene confers resistance to chloramphenicol. Identity region between λ and Dlp12 (A) spans across essential lysis genes (R and Rz) and terminase genes (A and nu1), separated by the <i>cos</i> site. Identity regions between λ and both Qin (B) and Rac (C) span across side tail fibers genes (<i>stf</i> and <i>tfa</i>). The <i>ble</i> gene, that confers resistance to phleomycine, was inserted between <i>tfa</i> and <i>ea47</i>, under the constitutive promoter P<i>sacB</i>. Blue arrows in C indicate the position of the 3 oligonucleotides used to sequence the recombinants. D: Schematic representation of the recombination assay: (1) λ phage is multiplied on a strain in which an antibiotic cassette has been inserted in a region of homology. (2) The phage produced is used to lysogenize a new strain. The total number of lysogenized bacteria is determined by their phleomycine resistance, while bacteria lyzogenised by a recombinant phage are also resistant to either chloramphenicol or kanamycin. E: Frequency of λ recombinants with Dlp12, Qin and Rac. Bp numbers indicate the size of the homology regions. Mean ± standard deviation of at least 3 independent recombination assays is indicated.</p

Table_1_Sak4 of Phage HK620 Is a RecA Remote Homolog With Single-Strand Annealing Activity Stimulated by Its Cognate SSB Protein.DOCX

<p>Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.</p

Table_6_Sak4 of Phage HK620 Is a RecA Remote Homolog With Single-Strand Annealing Activity Stimulated by Its Cognate SSB Protein.DOCX

<p>Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.</p

Presentation_1_Sak4 of Phage HK620 Is a RecA Remote Homolog With Single-Strand Annealing Activity Stimulated by Its Cognate SSB Protein.PDF

<p>Bacteriophages are remarkable for the wide diversity of proteins they encode to perform DNA replication and homologous recombination. Looking back at these ancestral forms of life may help understanding how similar proteins work in more sophisticated organisms. For instance, the Sak4 family is composed of proteins similar to the archaeal RadB protein, a Rad51 paralog. We have previously shown that Sak4 allowed single-strand annealing in vivo, but only weakly compared to the phage λ Redβ protein, highlighting putatively that Sak4 requires partners to be efficient. Here, we report that the purified Sak4 of phage HK620 infecting Escherichia coli is a poorly efficient annealase on its own. A distant homolog of SSB, which gene is usually next to the sak4 gene in various species of phages, highly stimulates its recombineering activity in vivo. In vitro, Sak4 binds single-stranded DNA and performs single-strand annealing in an ATP-dependent way. Remarkably, the single-strand annealing activity of Sak4 is stimulated by its cognate SSB. The last six C-terminal amino acids of this SSB are essential for the binding of Sak4 to SSB-covered single-stranded DNA, as well as for the stimulation of its annealase activity. Finally, expression of sak4 and ssb from HK620 can promote low-level of recombination in vivo, though Sak4 and its SSB are unable to promote strand exchange in vitro. Regarding its homology with RecA, Sak4 could represent a link between two previously distinct types of recombinases, i.e., annealases that help strand exchange proteins and strand exchange proteins themselves.</p