The site-specific integration reaction of Listeria phage A118 integrase, a serine recombinase

Abstract

BackgroundA large subfamily of serine recombinases contains long polypeptide segments appended to the C-terminal end of the conserved catalytic domain. Members of this subfamily often function as phage integrases but also mediate transposition and regulate terminal differentiation processes in eubacteria. Although a few members of this subfamily have been studied in purified in vitro systems, key mechanistic aspects of reactions promoted by these recombinases remain to be determined, particularly with respect to the functions of the large C-terminal domain.ResultsWe have developed and characterized a robust in vitro recombination reaction by the Listeria phage A118 integrase, a member of the subfamily of serine recombinases containing a large C-terminal domain. The reaction occurs in a simple buffered salt solution and exhibits a modest stimulation by divalent cations or spermidine and DNA supercoiling. Recombination with purified A118 integrase is unidirectional, being efficient only between attP and attB DNA sites to either join separate DNA molecules (intermolecular recombination) or to generate deletions or inversions depending on the relative orientation of att sites in cis (intramolecular recombination). The minimal attP site is 50 bp but requires only 44 bp of base sequence information, whereas the minimal attB site is 42 bp and requires 38 bp of base sequence information. DNA exchange occurs between the central 2 bp of attP and attB. Identity between these two base pairs is required for recombination, and they solely determine the orientation of recombination sites. The integrase dimer binds efficiently to full att sites, including the attL and attR integration products, but poorly and differentially to each half-site. The large C-terminal domain can be separated from the N-terminal catalytic by partial proteolysis and mediates non-cooperative DNA binding to att sites.ConclusionsThe basic properties of the phage A118 integrase reaction and its substrate requirements have been elucidated. A118 integrase thus joins the handful of biochemically characterized serine integrases that are serving as models for mechanistic studies on this important class of recombinases. Information reported here will also be useful in exploiting this recombinase for genetic engineering.

Control of Recombination Directionality by the Listeria Phage A118 Protein Gp44 and the Coiled-Coil Motif of Its Serine Integrase

“…Fully ligated relaxed circular products from processive DNA exchanges are also efficiently generated during A118 Int-catalyzed BP reactions on substrates in which the central 2 bp at the crossover sites are not equivalent (not shown). In addition, ethidium bromide gels reveal relaxed ligated circular products in wild-type BP reactions at early reaction times (not shown) that are consistent with the products from antiparallel synapses observed previously (23).…”

“…We showed previously that the monomeric Int-CTD binds with very different affinities to the four individual att half sites and noncooperatively to att full sites, unlike full-length Int (23). We asked how Gp44 influences Int-CTD binding to the different att full sites.…”

“…Previous studies showed that Int alone promoted only BP and weak PP recombination reactions (23,24). We evaluated the effect of Gp44 on noncognate partner recombination using the more stringent intermolecular reactions between supercoiled plasmids containing attL or attR and linear DNA fragments containing each of the four att sites.…”

“…The monomeric C-terminal domain (CTD; residues 148 to 452) (Fig. 4A) (23,24) was substituted for the full-length integrase and was found to also be sufficient for interaction with Gp44 (Fig. 4B).…”

“…We have been studying the phage A118 serine integrase from Listeria monocytogenes (23,24). A118 integrates into the comK gene within the L. monocytogenes chromosome by recombination between the phage attP and chromosome attB loci to generate the hybrid sites attL and attR (25).…”