Bacteriophage mv4 site-specific recombination: the central role of the P2 mv4Int-binding site

Abstract

The 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.

FIG 1

(A) Structure and nucleotide sequence of the mv4 attP region. The core sequence is underlined. 0 corresponds to the central base of the core region. The black arrows indicate the 11-bp consensus sequence 5′-TRRYTRRWARR-3′, which is present in five copies in mv4 attP and corresponds to the Int arm-type binding sites. The gray arrows indicate the 6-bp sequence of the Xis-binding site. The mutations in the Int arm-type binding sites are indicated above the nucleotide sequence. (B) Structure and nucleotide sequence of the P2-core region. The 17-bp core sequence and the P2 Int arm-type binding site are indicated by a light gray box and a dark gray box, respectively. The gray bent arrows indicate the 16-bp minimal attB site (attB^min). The dark vertical arrows indicate the strand exchange loci on the top and bottom strands. The dashed box encircles the overlap region.

FIG 2

In vitro site-specific recombination. Effects of mutations of the binding sites on integrative (A) and excisive (B and C) recombinations are depicted. The efficiency of recombination between supercoiled plasmid bearing attP or attR or attL (200 ng [0.1 pmol]) and linearly radiolabeled attB or attL or attR (10⁴ cpm) in the presence of ^mv4Int-enriched cell extract (3 μg [∼20 pmol]), with or without Xis cell extracts (3 μg [∼10 pmol]), was quantified as previously described (7). The values reported in the histograms are means and standard deviations of the results of 4 independent experiments.

FIG 3

Identification of the positions of strand exchange. (A) Localization of the position of the cleavage on the top strand. The result of the in vitro recombination assay on the suicide substrate (9) was analyzed on a 12% acrylamide–7 M urea gel in parallel with a sequence scale. At the top of the gel, the suicide substrate is schematized. In lanes T, the reaction mixture contained only the radiolabeled substrate without cell extract. In lane ^mv4Int, the reaction mixture contained the radiolabeled substrate in the presence of an ^mv4Int-enriched cell extract (3 μg [∼20 pmol]). The arrow indicates the length of the cleaved fragment. The sequence of the suicide substrate is indicated below the autoradiogram. (B) Localization of the position of the cleavage on the bottom strand. The reactions were analyzed as described for panel A.