Control of Directionality in Streptomyces Phage φBT1 Integrase-Mediated Site-Specific Recombination

Abstract

Streptomyces phage φBT1 integrates its genome into the attB site of the host chromosome with the attP site to generate attL and attR. The φBT1 integrase belongs to the large serine recombinase subfamily which directly binds to target sites to initiate double strand breakage and exchange. A recombination directionality factor (RDF) is commonly required for switching integration to excision. Here we report the characterization of the RDF protein for φBT1 recombination. The RDF, is a phage-encoded gp3 gene product (28 KDa), which allows efficient active excision between attL and attR, and inhibits integration between attB and attP; Gp3 can also catalyze topological relaxation with the integrase of supercoiled plasmids containing a single excision site. Further study showed that Gp3 could form a dimer and interact with the integrase whether it bound to the substrate or not. The synapse formation of attL or attR alone with integrase and Gp3 showed that synapsis did not discriminate between the two sites, indicating that complementarity of central dinucleotides is the sole determinant of outcome in correct excision synapses. Furthermore, both in vitro and in vivo evidence support that the RDFs of φBT1 and φC31 were fully exchangeable, despite the low amino acid sequence identity of the two integrases.

The Complete Genome Sequence of Escherichia coli EC958: A High Quality Reference Sequence for the Globally Disseminated Multidrug Resistant E. coli O25b:H4-ST131 Clone

“…PacBio thus offers a novel solution for studying the mechanism of phage tail fibre switching, and more generally, for the function of DNA invertase and other site-specific recombinases. For example, the DNA invertase gene has been severely truncated in the Phi4 prophage, suggesting that the inversion observed in this study must have been mediated by another enzyme in trans , as has been previously reported [49]–[51]. Notably, the Phi1 and Phi4 prophages encode near-identical 26 bp crossover sites at either end of their respective invertible segments (Table 3), suggesting that the Phi1 DNA invertase may be capable of mediating inversion at heterologous sites within the Phi4 prophage.…”

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

“…S1 in the supplemental material), but they do not generate recombinant products presumably because the core (crossover) nucleotides are not complementary after odd numbers of subunit rotations, which prevents ligation. The ability of the RDF to enhance a more promiscuous reaction seems to be a general property of serine integrases, as it has been also documented in the Bxb1, Rv1, C31, and Bt1 systems (8,9,38). As elaborated below, the RDFs may remodel Int to both disrupt an inhibitory conformation and to directly promote specific synaptic complex architectures by orienting the trajectory of the CC motif.…”

“…1B). It is likely that Gp44 has additional roles in A118 biology, as has been described for other RDFs (9,(40)(41)(42).…”

“…Alternatively, higher levels of Gp44 may be required to unlock competitive intramolecular coiled-coil interactions in the LR reaction that are not present in the Int-bound attP and attB sites. Where evaluated, the large RDFs for serine integrases appear to behave similarly with respect to relative stoichiometries for LR excision versus BP inhibition (7)(8)(9). However, the various RDFs behave differently with respect to their apparent affinities for and resulting electrophoretic migration changes to the four att-Int complexes.…”

“…Many phage integrases are members of a subfamily of serine recombinases (SRs) that are known as large SRs due to their very long C-terminal DNA binding regions relative to other SRs (1)(2)(3)(4). In the few serine integrase systems that have been studied, phage-encoded recombination directionality factors (RDFs) that function together with their cognate integrases control the direction of the recombination reaction, integration or excision, and thus perform an essential function in the developmental pathways of lysogenic phage (5)(6)(7)(8)(9). The RDFs of serine integrases that have been studied thus far function mechanistically differently than the RDFs (or Xis) proteins of tyrosine integrases, such as those from phages , HP1, P2, P22, and L5, which are largely DNA architectural proteins (10)(11)(12)(13)(14).…”