The Structure of the Excisionase (Xis) Protein from Conjugative Transposon Tn916 Provides Insights into the Regulation of Heterobivalent Tyrosine Recombinases

Abbani, Mohamad A.; Iwahara, Mizuho; Clubb, Robert T.

doi:10.1016/j.jmb.2005.01.019

Cited by 21 publications

(21 citation statements)

References 85 publications

(75 reference statements)

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“…8). The large area of protection identified by the DNase I footprint, in agreement with EMSA data, suggests that several Xis subunits may interact specifically with identified regions of attP DNA or may possibly bend or loop the DNA as has been observed for the IHF protein (28,29), the Tn916 Xis protein (1,30), and the lambda Xis protein (2,25,38).…”

Section: Discussionsupporting

confidence: 80%

Interaction of the Gifsy-1 Xis Protein with the Gifsy-1 attP Sequence

Flanigan

Gardner

2007

J Bacteriol

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The Gifsy-1 phage integrates site specifically into the Salmonella chromosome via an integrase-mediated site-specific recombination mechanism. Initial genetic analysis suggests that Gifsy-1 integrase-mediated excision of the Gifsy-1 phage is influenced by proteins encoded by both the Gifsy-1 and the Gifsy-2 phages. Our studies show that the Gifsy-1 Xis protein regulates the directionality of integrase-mediated excision of the Gifsy-1 phage. Electrophoretic mobility shift assays, DNase I footprinting, dimethyl sulfate (DMS) interference assays, and DMS protection assays were used to identify a 31-base-pair sequence in the attP region to which the Gifsy-1 protein binds. The results suggest that this recombination directionality factor binds in vitro to three imperfect direct repeats, spaced 10 base pairs apart, in a sequential and cooperative manner in the absence of other phage-encoded proteins. Our studies suggest that, while the Gifsy-1 Xis does not require additional factors for specific and high-affinity binding, it may form a microfilament on DNA similar to that described for the phage lambda Xis protein.

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Section: Discussionsupporting

confidence: 80%

Interaction of the Gifsy-1 Xis Protein with the Gifsy-1 attP Sequence

Flanigan

Gardner

2007

J Bacteriol

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“…The importance of integrase stability has also been demonstrated in the excision of a mycophage (which does not appear to require an RDF for excision) (54). The majority of RDFs that have been described are from prophages and were shown to control directionality of recombination by playing an architectural role by binding at the att sites, to each other, and/or to the integrase (20,21,24,(27)(28)(29)55). We determined that VefA and VefB bound the att sites of both islands, although VefB bound both sites at a higher affinity, causing a shift with four times less protein than VefA.…”

Section: Discussionmentioning

confidence: 99%

Pathogenicity Island Cross Talk Mediated by Recombination Directionality Factors Facilitates Excision from the Chromosome

2016

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Pathogenicity islands (PAIs) are mobile integrated genetic elements (MIGEs) that contain a diverse range of virulence factors and are essential in the evolution of pathogenic bacteria. PAIs are widespread among bacteria and integrate into the host genome, commonly at a tRNA locus, via integrase-mediated site-specific recombination. The excision of PAIs is the first step in the horizontal transfer of these elements and is not well understood. In this study, we examined the role of recombination directionality factors (RDFs) and their relationship with integrases in the excision of two PAIs essential for Vibrio cholerae host colonization: Vibrio pathogenicity island 1 (VPI-1) and VPI-2. VPI-1 does not contain an RDF, which allowed us to answer the question of whether RDFs are an absolute requirement for excision. We found that an RDF was required for efficient excision of VPI-2 but not VPI-1 and that RDFs can induce excision of both islands. Expression data revealed that the RDFs act as transcriptional repressors to both VPI-1-and VPI-2-encoded integrases. We demonstrated that the RDFs Vibrio excision factor A (VefA) and VefB bind at the attachment sites (overlapping the int promoter region) of VPI-1 and VPI-2, thus supporting this mode of integrase repression. In addition, V. cholerae RDFs are promiscuous due to their dual functions of promoting excision of both VPI-1 and VPI-2 and acting as negative transcriptional regulators of the integrases. This is the first demonstration of cross talk between PAIs mediated via RDFs which reveals the complex interactions that occur between separately acquired MIGEs. IMPORTANCEDeciphering the mechanisms of pathogenicity island excision is necessary for understanding the evolution and spread of these elements to their nonpathogenic counterparts. Such mechanistic insight would assist in predicting the mobility of uncharacterized genetic elements. This study identified extensive RDF-mediated cross talk between two nonhomologous VPIs and demonstrated the dual functionality of RDF proteins: (i) inducing PAI excision and (ii) acting as transcriptional regulators. Findings from this study may be implicated in determining the mobilome contribution of other bacteria with multiple MIGEs. Mobile integrative genetic elements (MIGEs) are the vectors of horizontal gene transfer (HGT) among bacteria via the mechanisms of transformation, transduction, and conjugation (1-3). Members of MIGEs include transposons, integrons, bacteriophages, plasmids, and integrative conjugative elements (ICEs)/ conjugative transposons and genomic/pathogenicity islands (GEIs/PAIs). Commensal bacteria can be converted into deadly pathogens through HGT of MIGEs that contain virulence factors, or a pathogen may become more virulent with the acquisition of additional virulence factors (1, 4-6). PAIs were first described for uropathogenic Escherichia coli (UPEC) and have since been described for many pathogenic bacteria (7-17). PAIs range in size from 10 to 200 kb and have a guanine and cytosine (GC) content ...

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“…Xis is the founding member of a diverse family of proteins that function to control reactions that rearrange DNA, called recombination directionality factors (RDFs) (24). Many of the RDFs share two common features with Xis: they adopt a similar winged-helix fold and cooperatively bind DNA as oligomers to produce extensive footprints (25)(26)(27)(28)(29)(30). For example, the small Xis protein encoded by mycobacteriophage L5 is predicted to bind to four related sequence motifs spaced Ϸ10 bp apart to form a stable and bent DNA complex (27).…”

Section: The Structure Of Three Xis Proteins Bound To the X1-x2 Dna Smentioning

confidence: 99%

Structure of the cooperative Xis–DNA complex reveals a micronucleoprotein filament that regulates phage lambda intasome assembly

Abbani¹,

Papagiannis

Sam³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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The DNA architectural protein Xis regulates the construction of higher-order nucleoprotein intasomes that integrate and excise the genome of phage lambda from the Escherichia coli chromosome. Xis modulates the directionality of site-specific recombination by stimulating phage excision 10 6 -fold, while simultaneously inhibiting phage reintegration. Control is exerted by cooperatively assembling onto a Ϸ35-bp DNA regulatory element, which it distorts to preferentially stabilize an excisive intasome. Here, we report the 2.6-Å crystal structure of the complex between three cooperatively bound Xis proteins and a 33-bp DNA containing the regulatory element. Xis binds DNA in a head-to-tail orientation to generate a micronucleoprotein filament. Although each protomer is anchored to the duplex by a similar set of nonbase specific contacts, malleable protein-DNA interactions enable binding to sites that differ in nucleotide sequence. Proteins at the ends of the duplex sequence specifically recognize similar binding sites and participate in cooperative binding via protein-protein interactions with a bridging Xis protomer that is bound in a less specific manner. Formation of this polymer introduces Ϸ72°of curvature into the DNA with slight positive writhe, which functions to connect disparate segments of DNA bridged by integrase within the excisive intasome.DNA bending ͉ recombination directionality factors ͉ site-specific DNA recombination ͉ x-ray structure M obile genetic elements such as bacteriophages, conjugative transposons, and pathogenicity islands promote the lateral exchange of foreign DNA, enabling bacteria to acquire metabolic, pathogenic, and antibiotic resistance determinants. To prevent potentially catastrophic changes in the genome, these DNA rearrangements are often tightly controlled by regulatory factors that function together with the recombinase. The integration and excision reactions of phage , which are controlled by the phageencoded Xis protein, serve as a paradigm for studies of regulated site-specific recombination (1). Upon infection, specific DNA attachment sites located on the circularized phage genome (attP) and bacterial chromosome (attB) recombine to generate the integrated prophage with flanking hybrid sites (attL and attR) (Fig. 1A). Cellular DNA damage initiates a series of events that result in prophage excision to regenerate attP on the episomal phage genome and attB on the chromosome. Although the DNA strand transfer steps in each reaction are catalyzed by the phage-encoded tyrosine recombinase integrase (Int) protein, and are mechanistically similar, directionality control is achieved by guiding the assembly of distinct higher-order nucleoprotein structures called intasomes. Viral integration occurs within an integrative intasome containing Int and the Escherichia coli-encoded integration host factor (IHF) (2, 3), whereas excision is performed within an alternative excisive intasome complex containing Int, Xis, IHF, and the factor for inversion stimulation (4-7).Xis is the master regu...

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The Structure of the Excisionase (Xis) Protein from Conjugative Transposon Tn916 Provides Insights into the Regulation of Heterobivalent Tyrosine Recombinases

Cited by 21 publications

References 85 publications

Interaction of the Gifsy-1 Xis Protein with the Gifsy-1 attP Sequence

Interaction of the Gifsy-1 Xis Protein with the Gifsy-1 attP Sequence

Pathogenicity Island Cross Talk Mediated by Recombination Directionality Factors Facilitates Excision from the Chromosome

Structure of the cooperative Xis–DNA complex reveals a micronucleoprotein filament that regulates phage lambda intasome assembly

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