A phage protein that binds φC31 integrase to switch its directionality

Khaleel, Thanafez; Younger, Ellen; McEwan, Andrew R.; Varghese, Anpu S.; Smith, Margaret C. M.

doi:10.1111/j.1365-2958.2011.07696.x

Cited by 99 publications

(154 citation statements)

References 40 publications

Supporting

Mentioning

141

Contrasting

Order By: Relevance

“…3E, the reactions are most efficient when Int plus Gp44 are preincubated together in solution prior to the addition of substrate DNA. This feature has been observed for Gp3-activated LR reactions by the C31 integrase (8) and is consistent with the RDF remodeling its cognate integrase into a more active conformation. When added, Gp44 was preincubated with Int for most of the experiments described in this paper, including the DNA binding reactions described below, which are also enhanced by preincubation.…”

Section: Resultssupporting

confidence: 86%

“…An alternative explanation is that the plateau behavior reflects an equilibrium state generated by reactions back to the attL and attR parental forms under limiting Gp44 concentrations. Stoichiometric requirements of the RDF for the excision reaction by Bxb1 and C31 integrases have also been reported (7,8,37). There is a demonstrable effect by the order of addition of components to LR reactions.…”

Section: Resultsmentioning

confidence: 85%

“…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).…”

mentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2017

View full text Add to dashboard Cite

The serine integrase of phage A118 catalyzes integrative recombination between attP on the phage and a specific attB locus on the chromosome of Listeria monocytogenes, but it is unable to promote excisive recombination between the hybrid attL and attR sites found on the integrated prophage without assistance by a recombination directionality factor (RDF). We have identified and characterized the phage-encoded RDF Gp44, which activates the A118 integrase for excision and inhibits integration. Gp44 binds to the C-terminal DNA binding domain of integrase, and we have localized the primary binding site to be within the mobile coiled-coil (CC) motif but distinct from the distal tip of the CC that is required for recombination. This interaction is sufficient to inhibit integration, but a second interaction involving the N-terminal end of Gp44 is also required to activate excision. We provide evidence that these two contacts modulate the trajectory of the CC motifs as they extend out from the integrase core in a manner dependent upon the identities of the four att sites. Our results support a model whereby Gp44 shapes the Int-bound complexes to control which att sites can synapse and recombine.IMPORTANCE Serine integrases mediate directional recombination between bacteriophage and bacterial chromosomes. These highly regulated site-specific recombination reactions are integral to the life cycle of temperate phage and, in the case of Listeria monocytogenes lysogenized by A118 family phage, are an essential virulence determinant. Serine integrases are also utilized as tools for genetic engineering and synthetic biology because of their exquisite unidirectional control of the DNA exchange reaction. Here, we identify and characterize the recombination directionality factor (RDF) that activates excision and inhibits integration reactions by the phage A118 integrase. We provide evidence that the A118 RDF binds to and modulates the trajectory of the long coiled-coil motif that extends from the large carboxyl-terminal DNA binding domain and is postulated to control the early steps of recombination site synapsis.KEYWORDS site-specific DNA recombination, serine integrase, recombination directionality factor, Listeria monocytogenes T emperate phage genomes integrate into and excise out of bacterial chromosomes using phage-specific site-specific recombination systems. 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 (1-4). In the few serine integrase systems that have been studied, phage-encoded recombination

show abstract

Section: Resultssupporting

confidence: 86%

Section: Resultsmentioning

confidence: 85%

mentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2017

View full text Add to dashboard Cite

show abstract

“…The intergenic regions to the right of the int genes are only moderately conserved between Rey and the subcluster M1 phages, raising the question as to whether they use the same attB site. Predicting the location of a putative recombination directionality factor (RDF) is complicated by the variety of different proteins that can perform this function (44)(45)(46). However, we note that located near the integrases are genes encoding phosphoesterases (Bongo gp125, PegLeg gp129, Rey gp144), reminiscent of the RDF encoded by the gene for Bxb1 (gp47) (45), which is also predicted to function as a phosphoesterase.…”

Section: Resultsmentioning

confidence: 98%

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotypes

Pope

Anders

Baird

et al. 2014

J Virol

View full text Add to dashboard Cite

Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc 2 155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCEThe bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis, provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.T he bacteriophage population is large, dynamic, old, and genetically diverse (1). Over 1,000 phage genomes have been sequenced, and the majority are double-stranded DNA (dsDNA) tailed phages, classified morphologically in the order Caudovirales. The genomes of dsDNA tailed phages vary in length from ϳ20 kbp to over 500 kbp (2) and typically contain 20 to 30 genes encoding virion structure and assembly functions, genes coding for DNA and nucleotide metabolism, a lysis cassette, and regulatory systems. The genomes of temperate phages typically encode repressors, contain operators, and frequently include systems for phage genome integration. However, with the exception of the few well-studied phage prototypes, the majority of genes carried by phages are of unknown function (1). Mycobacteriophages-vir...

show abstract

“…As a result, several φC31 integrase mutants made significant improvements in recombination efficiency and specificity (Liesner et al, 2006;Keravala et al, 2009). Moreover, some mutants gain the extra ability of excision (the reverse reaction of integration) McEwan et al, 2009), which normally takes place in the presence of a recombination directionality factor, gp3 protein (Khaleel et al, 2011), which provides us with a clue to explore the possible mechanism of φC31 integrase controlling the reaction direction. Here, we focus on the hyperactive mutants that are both active in integration and excision and then present a possible way in which φC31 integrase regulates the recombination direction.…”

Section: Introductionmentioning

confidence: 99%

A possible way that φC31 integrase regulates the recombination direction

et al. 2015

View full text Add to dashboard Cite

ABSTRACT. φC31 integrase encoded by Streptomyces phage can mediate site-specific recombination between phage and host genomes. The recombination direction is generally considered to be accurately regulated, but the regulatory mechanisms involved are still unclear. Recently, some hyperactive mutants of φC31 integrase that can bypass the regulatory steps have been isolated and extensively studied. A putative coiled-coil region is found to play a critical role in controlling recombination direction. Further analysis led us to the speculation that at least two regions in the N-terminal domain of φC31 integrase are involved in the tetrameric interfaces and that the putative coiled coil interacts with one of the regions to regulate the recombination direction.

show abstract

A phage protein that binds φC31 integrase to switch its directionality

Cited by 99 publications

References 40 publications

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

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

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotypes

A possible way that φC31 integrase regulates the recombination direction

Contact Info

Product

Resources

About