Xis Protein Binding to the Left Arm Stimulates Excision of Conjugative Transposon Tn<i>916</i>

Connolly, Kevin M.; Iwahara, Mizuho; Clubb, Robert T.

doi:10.1128/jb.184.8.2088-2099.2002

Cited by 22 publications

(21 citation statements)

References 66 publications

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“…Thus, the himA mutation appears to have a modest effect on V. cholerae growth. These results are similar to what has been reported for fis and himA in E. coli (8,14,22).…”

Section: Resultssupporting

confidence: 82%

Requirement for Vibrio cholerae Integration Host Factor in Conjugative DNA Transfer

2006

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The requirement for host factors in the transmission of integrative and conjugative elements (ICEs) has not been extensively explored. Here we tested whether integration host factor (IHF) or Fis, two host-encoded nucleoid proteins, are required for transfer of SXT, a Vibrio cholerae-derived ICE that can be transmitted to many gram-negative species. Fis did not influence the transfer of SXT to or from V. cholerae. In contrast, IHF proved to be required for V. cholerae to act as an SXT donor. In the absence of IHF, V. cholerae displayed a modest defect for serving as an SXT recipient. Surprisingly, SXT integration into or excision from the V. cholerae chromosome, which requires an SXT-encoded integrase related to integrase, did not require IHF. Therefore, the defect in SXT transmission in the V. cholerae IHF mutant is probably not related to IHF's ability to promote DNA recombination. The V. cholerae IHF mutant was also highly impaired as a donor of RP4, a broad-host-range conjugative plasmid. Thus, the V. cholerae IHF mutant appears to have a general defect in conjugation. Escherichia coli IHF mutants were not impaired as donors or recipients of SXT or RP4, indicating that IHF is a V. cholerae-specific conjugation factor.

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“…Thus, the himA mutation appears to have a modest effect on V. cholerae growth. These results are similar to what has been reported for fis and himA in E. coli (8,14,22).…”

Section: Resultssupporting

confidence: 82%

Requirement for Vibrio cholerae Integration Host Factor in Conjugative DNA Transfer

2006

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“…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). The low molecular weight Xis protein from the Tn916 transposon also exhibits similar binding behavior and produces large footprints that contain hypersensitive cleavage sites characteristic of wrapped or bent DNA (31,32). It seems likely that many of these proteins also assemble into nucleoprotein filaments, which function to stabilize recombinogenic higher-order structures.…”

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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“…2). DnaseI protection assays with Xis show that it binds to a region which exceeds what was expected based on the size of the protein [1,5]. This can be explained by postulating that DNA is wrapped around Xis, thus protecting a larger region.…”

Section: Functional Analysis Of Int and Xismentioning

confidence: 79%

“…Another possibility is that Xis may bind to the DNA in the crook of a 2018 P. Mullany, A. P. Roberts and H. Wang Integration and excision static bend. Therefore, Xis may facilitate the binding of Int to DNA by bending the target DNA (but see below for further discussion of this point) or by formation of proteinprotein or protein-DNA complexes, or both [1,5]. Recent work has shown that mutations at the right end of Tn916 that decrease the binding of Xis result in a higher frequency of excision compared with the wild type, indicating that Xis inhibits excision [1].…”

Section: Functional Analysis Of Int and Xismentioning

confidence: 95%

“…However, in l Xis facilitates the action of Int by bending the DNA to allow the C-terminal domain of Int to bind to phage DNA. In the Tn916 system the Nand C-terminal domains of Int are large enough to allow DNA bending to occur without the help of another protein [5]. Therefore, Xis may exert its effect via protein-protein interactions.…”

Section: Functional Analysis Of Int and Xismentioning

confidence: 98%

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Mechanism of integration and excision in conjugative transposons

Mullany¹,

Roberts²,

Wang³

2002

Cellular and Molecular Life Sciences (CMLS)

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Xis Protein Binding to the Left Arm Stimulates Excision of Conjugative Transposon Tn916

Cited by 22 publications

References 66 publications

Requirement for Vibrio cholerae Integration Host Factor in Conjugative DNA Transfer

Requirement for Vibrio cholerae Integration Host Factor in Conjugative DNA Transfer

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

Mechanism of integration and excision in conjugative transposons

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