Differential Affinity and Cooperativity Functions of the Amino-terminal 70 Residues of λ Integrase

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Lambda integrase (Int) is a heterobivalent DNA-binding protein that together with the accessory DNA-bending proteins IHF, Fis, and Xis, forms the higher-order protein-DNA complexes that execute integrative and excisive recombination at specific loci on the chromosomes of phage and its Escherichia coli host. The large carboxyl-terminal domain of Int is responsible for binding to core-type DNA sites and catalysis of DNA cleavage and ligation reactions. The small amino-terminal domain (residues 1-70), which specifies binding to arm-type DNA sites distant from the regions of strand exchange, consists of a three-stranded ␤-sheet, proposed to recognize the cognate DNA site, and an ␣-helix. We report here that a site on this ␣-helix is critical for both homomeric interactions between Int protomers and heteromeric interactions with Xis. The mutant E47A, which was identified by alanine-scanning mutagenesis, abolishes interactions between Int and Xis bound at adjacent binding sites and reduces interactions between Int protomers bound at adjacent arm-type sites. Concomitantly, this residue is essential for excisive recombination and contributes to the efficiency of the integrative reaction. NMR titration data with a peptide corresponding to Xis residues 57-69 strongly suggest that the carboxyl-terminal tail of Xis and the ␣-helix of the aminoterminal domain of Int comprise the primary interaction surface for these two proteins. The use of a common site on Int for both homotypic and heterotypic interactions fits well with the complex regulatory patterns associated with this site-specific recombination reaction.T he bacteriophage -encoded integrase (Int) belongs to a subgroup of the tyrosine recombinase family known as the heterobivalent recombinases (1, 2). These recombinases catalyze reactions that are distinguished from other pathways of sitespecific rearrangement and movement of DNA by their directionality. This feature is built on the ability of these recombinases to simultaneously bind and bridge two distinct and well separated DNA-binding sites (3-5). Int binds with high affinity to arm-type DNA sites by means of a small amino-terminal domain (residues 1-70) whereas it binds with lower affinity to core-type sites by means of a larger carboxyl-terminal domain (residues 75-356), which also executes DNA strand cleavage and ligation. Bridging between arm and core sites is facilitated by DNA bends induced by accessory proteins (IHF, Xis, and Fis) bound to DNA sites located between the arm-and core-type sequences (5-9). This ensemble of DNA-bending-and DNA-bridging-proteins forms the large recombinogenic complexes that confer directionality on the recombination reaction. In this article we show that formation of both the Int-Xis and Int-Int complexes, key elements in the structure and function of the higher-order recombinogenic structures, depends on a common site in the amino-terminal domain of Int.The Int recombinase catalyzes the integration and excision of viral DNA into and out of the chromosome of its Escherichia coli...

Section: Resultssupporting

confidence: 90%

“…This procedure aided purification and eliminated complications from protein-protein interactions known to be associated with the much larger carboxyl-terminal domain. The N1-75 fragment has already been shown to be a good model for the amino-terminal functions of full-length Int (40).…”

Section: Resultsmentioning

confidence: 99%

Identification of the λ integrase surface that interacts with Xis reveals a residue that is also critical for Int dimer formation

Warren

Sam²,

Manley³

et al. 2003

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“…Consequently the P-arm would not be properly positioned to stabilize a synapsiscompetent attR. Because Xis also promotes cooperative binding of Int at P2 (37)(38)(39), it seems likely that only low-affinity Int-attR interactions would be possible in its absence.…”

Section: Discussionmentioning

confidence: 99%

Nucleoprotein architectures regulating the directionality of viral integration and excision

Seah

Warren

Tong

et al. 2014

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The virally encoded site-specific recombinase Int collaborates with its accessory DNA bending proteins IHF, Xis, and Fis to assemble two distinct, very large, nucleoprotein complexes that carry out either integrative or excisive recombination along regulated and essentially unidirectional pathways. The core of each complex consists of a tetramer of Integrase protein (Int), which is a heterobivalent DNA binding protein that binds and bridges a core-type DNA site (where strand cleavage and ligation are executed), and a distal arm-type site, that is brought within range by one or more DNA bending proteins. The recent determination of the patterns of these Int bridges has made it possible to think realistically about the global architecture of the recombinogenic complexes. Here, we combined the previously determined Int bridging patterns with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein Holiday junction recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination. Our confidence in the basic features of these architectures is based on the redundancy and self-consistency of the underlying data from two very different experimental approaches to establish bridging interactions, a set of strategic intracomplex distances from FRET experiments, and the model's ability to explain key aspects of the integrative and excisive recombination pathways, such as topological changes, the mechanism of capturing attB, and the features of asymmetry and flexibility within the complexes.regulation of directionality | topology | recombinogenic architectures | molecular machines H igh-precision DNA transactions responsible for a variety of fundamental processes are typically promoted and modulated by large multiprotein machines that use cooperative interactions and involve DNA bending and/or wrapping. One well-studied example is the tightly regulated and highly directional site-specific recombination by which bacteriophage λ inserts and excises its DNA into and out of the Escherichia coli host chromosome, using the phage attP and the bacterial attB DNA sequences for integration and the resulting junction sequences, attL and attR, for excision. These reactions are catalyzed by the phage-encoded Integrase protein (Int), the founding member of the tyrosine recombinase family of site-specific recombinases (1). In addition to mediating integration and excision of viral genomes, members of this family function in a variety of other cellular processes including chromosome segregation, gene regulation, and conjugative transposition (2).Int has three well-characterized domains: an N-terminal DNA-binding domain (NTD), a central core-binding domain (CB), and a C-terminal catalytic domain (CAT). The CB and CAT domains (referred to here as the CTD) are together responsible for binding to the core-type DNA sequences where strand exchange and ligation t...

“…6 and text below). Isolated PЈ1,2 arm DNA is bent Ϸ45-55°when bound by adjacent Int protomers, and it is also bent within the ternary complexes (11,17,37). This study indicates that, within the ternary complexes, this bend is probably Ͻ40°(the limit detectable by FRET) because we did not observe a decrease in the end-to-end distance of the arm DNA in the ternary complex compared with unbound arm DNA ( Table 1).…”

Section: Discussionmentioning

confidence: 54%

“…By means of a small N-terminal domain (residues 1-70), Int binds with high affinity to ''arm-type'' DNA sites that are distant from loweraffinity core-type sites where the C-terminal domain (residues 75-365) binds and executes DNA strand cleavage and ligation (9)(10)(11). The Int-mediated bridging is assisted by DNA bends induced by accessory proteins (IHF, Xis, and Fis) bound to sites located between the arm and core sequences.…”

mentioning

confidence: 99%

Architecture of recombination intermediates visualized by in-gel FRET of λ integrase–Holliday junction–arm DNA complexes

Radman‐Livaja

Biswas

Mierke

et al. 2005

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