Control of the serine integrase reaction: roles of the coiled-coil and helix E regions in DNA site synapsis and recombination

Abstract

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn, directs the relative trajectories of the CC motifs on each subunit of the att -bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal length CC motifs and 14 residues surrounding the tip where pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggests that molecular interactions between CC motif tips may differ in integrative ( attP x attB ) and excisive ( attL x attR ) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminantly, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after initial assembly of the integrase synaptic tetramer. Importance The robust and exquisitely-regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage, and in the case of the A118 prophage, are an important virulence factor by Listeria monocytogenes . The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

A phage weaponizes a satellite recombinase to subvert viral restriction

“…However, we observed that while Int maintained its recombinase activity in vitro in the presence of Adi, there was no obvious aberrant nuclease activity when the purified proteins were combined (Figure S5). Adi also did not inhibit the integration reaction as an RDF would ( 41 , 44 , 45 ), further confirming it is not the RDF. The inability to reconstitute destructive nuclease activity in vitro could be due to a missing factor (e.g.…”

A phage weaponizes a satellite recombinase to subvert viral restriction

“…However, we observed that while Int maintained its recombinase activity in vitro in the presence of Adi, there was no obvious aberrant nuclease activity when the purified proteins were combined (Figure S5). Adi also did not inhibit the integration reaction as an RDF would (41,44,45), further confirming it is not the RDF. The inability to reconstitute destructive nuclease activity in vitro could be due to a missing factor (e.g., other proteins or cofactors in the nuclease buffer).…”

“…Lastly, another way in which Int activity can be modulated is through modification of the protein itself. However, we are not aware of any LSRs that can undergo modification, except during recombination in which the LSR is covalently bound to the 5’ DNA ends through a phosphoseryl linkage (26, 39–41). Thus, further study of the mechanism by which Adi exploits Int activity to target PLE2 may reveal more widespread modifications to LSR activity than are currently appreciated.…”

“…Previous work has shown that the catalytic activity of LSRs is decoupled from DNA binding activity (26,(39)(40)(41). Therefore, we considered the possibility that Adi may exploit the DNA binding activity of Int to localize to attP and target it for degradation.…”

High fidelity one‐pot DNA assembly using orthogonal serine integrases

“…It is still unclear to what extent natural attP and attB sequences are optimal for LSI activity, [ 39–45 ] and it is likely that phages integrate into non‐optimal attB sites in some organisms. Recent studies have suggested ways through which the att sites of LSIs could be redesigned for improved activities by changing the spacing between DNA sections of the att site recognized by specific integrase domains, [ 41,46,47 ] but the universality of these ideas is yet to be tested across a wide range of LSIs. Also, optimization of reaction buffer might be needed for each LSI.…”