Bacteriophage mv4 Site-Specific Recombination: the Central Role of the P2 ^mv4 Int-Binding Site

Abstract

bThe contributions of the five mv4 Int-and two mv4 Xis arm-binding sites to the spatial intasome organization of bacteriophage mv4 were found not to be equivalent. The 8-bp overlap region was mapped to the left extremity of the core region and is directly flanked by the P2 Int arm-binding site. These results and the absence of characteristic Int core-binding sites suggest that the P2 site is the determinant for integrase positioning and recognition of the core region. During the establishment of the lysogeny, many temperate bacteriophages integrate their genome into the host bacterial chromosome through a site-specific recombination mechanism. This integration is catalyzed by a phage-encoded recombinase (integrase) and occurs between the attB and attP attachment sites located on the bacterial and phage genomes, respectively. Prophage excision during the induction of lytic growth is also catalyzed by the recombinase: it involves recombination between the attR and attL attachment junctions and requires at least one phage-encoded recombination directionality factor (RDF) in addition to the integrase.In well-characterized site-specific recombination systems, the protein binding sites at the att recombination sites are not all used in the same way for integrative or excisive recombination (1-3). The integrase of phage is a heterobivalent recombinase that binds two different families of DNA sequences: the "arm-type" and the "core-type" binding sites (4). The set of proteins and the number and combination of the binding sites used are different in integrative and excisive recombination reactions. During integrative recombination, in the intasome, Int binds to four arm-type binding sites (P1, P=1, P=2, and P=3) on attP whereas the accessory factor IHF binds to all three of its cognate sites (2). During excisive recombination, Xis binds to the X1, X1.5, and X2 sites on attR and Int to three of the arm-type sites (P2 on attR and P=1 and P=2 on attL) (2). In contrast, in phage KplE1, the same combination of IntS arm-type binding sites is used for both excisive and integra-

Characterisation and reprogramming of bacteriophage mv4 integrase recombination specificity

“…Interestingly, when considering the consensus sequences rather than the WT sequences, the DNA motifs surrounding the overlap region exhibited complementarity, with purines enrichment at the left-side and pyrimidines at the right-side. Thus, in contrast to the previous study that concluded there might be an arm-type site adjacent to the overlap region (42), our analysis strongly supports the classical organization of heterobivalent YR recombination systems for the 21-bp attB and core-attP sites, with imperfect inverted-repeated patterns flanking each strand-exchange region (Figure 4C), and defining BOB' and COC' structures for attB and core-attP respectively, in accordance with the phage lambda terminology.…”

“…Given the organisational peculiarities of the mv4 attP core-region and attB site (40, 42), both sites underwent a comprehensive experimental reevaluation using a random DNA libraries based approach (59), with the rational that DNA positions encompassing these core regions should display constraints in nucleotide composition. Those DNA libraries, corresponding either to the attB site or to the core-region of the attP site, harboured a range of 7 to 10 randomized positions (Figure 2A).…”

“…Nucleotides outside the attB minimal site (40) are indicated in grey, and sequence identical between core- attP and attB are in bold. The atypical P2 arm-type binding site and strand-exchange (O) region (42) are indicated by horizontal and vertical black arrows, respectively. Random nucleotides are represented by “N” and nucleotides identical to the native sequences are symbolized by dashes.…”

“…The erroneous aminoacids of mv4 Int(427aa) are shown in red, and the catalytic domain’s seven conserved residues are highlighted in grey. ( C ) Comparative genetic organisation of lambda (adapted from (13)) and mv4 (adapted from (42)) attP and attB sites. Arm-binding and core-binding sites for the integrase are represented by blue and red rectangles, respectively.…”

“…The “ mv4 Int/ attP/attB ” system is able to drive recombination in a wide range of bacteria, including E. coli and various Gram-positive species (41), indicating that it does not rely on species-specific host-factor to promote recombination. Furthermore, both attP and attB sites have atypical structures (Figure 1C), with unusual location of mv4 Int arm-binding sites on the attP site (39, 42), no inverted-repeats core-binding sites at the 17-bp core- attP site, and a non-canonical 8-bp overlap sequence (42). As these differences strongly suggested alternative recombination mechanism, we reanalysed the organisation of both attB and attP sites, through in vitro recombination with various random DNA libraries and DNA-binding experiments.…”