Abstract
Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between ‘attachment sites’ in the phage (attP) and the host (attB). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase bound to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP × attB synaptic complexes to initiate recombination, and how attL × attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR. The results provide a structural framework for understanding, testing and engineering serine integrase function.
Highlights
The large serine recombinases (LSRs) are DNArearranging enzymes that are members of the serine recombinase superfamily [1,2]
LI integrase differs by 11 conservative amino acid changes (98% identical) from the bacteriophage A118 integrase [22] and has similar attachment site sequences (Supplementary Figure S1)
For self-association of attachment sites’ in the phage (attP) sites and attB sites, the arrangements of zinc ribbon domain (ZD) in the participating half-sites differ significantly from those in the attP Â attB complex. We suggest that these alternative arrangements do not permit interactions between CC motifs that coincide with formation of an activated catalytic domain tetramer that can undergo strand exchange
Summary
The large serine recombinases (LSRs) are DNArearranging enzymes that are members of the serine recombinase superfamily [1,2]. Many LSRs are bacteriophage integrases (often referred to as serine integrases) whose function is to integrate the phage genome into a host chromosome by using short specific DNA sequences, or ‘attachment sites’, in the virus (attP) and in the host (attB). There are no requirements for accessory proteins, auxiliary DNA sequences, specific DNA topologies or supercoiling, and the short (40–50 bp) attachment sites allow for simple manipulation of substrates. Because of these properties, the attP Â attB integration reaction can be performed with high specificity and directionality in inter- and intramolecular contexts, and in a variety of eukaryotic cells [8,9,10,11,12]. The bacteriophage jC31 integrase has shown promise for use in gene therapy applications [13]
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