The Structure of Xis Reveals the Basis for Filament Formation and Insight into DNA Bending within a Mycobacteriophage Intasome

Abstract

The Recombination Directionality Factor, Xis, is a DNA bending protein that determines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein-DNA architectures. Although the attachment site DNA of Mycobacteriophage Pukovnik is likely to contain four sites for Xis binding, Xis crystals contain five subunits in the asymmetric unit, four of which align into a Xis filament, and a fifth that is generated by an unusual domain swap. Extensive intersubunit contacts stabilize a bent filament-like arrangement with Xis monomers aligned head-to-tail. The structure implies a DNA bend of ~120°, which is in agreement with DNA bending measured in vitro. Formation of attR-containing intasomes requires only Int and Xis, distinguishing Pukovnik from lambda. Therefore, we conclude that in Pukovnik, Xis-induced DNA bending is sufficient to promote intramolecular Int-mediated bridges during intasome formation.

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

“…The shift of the protein-bound DNA changes as the amount of protein increases, suggesting multiple Vef binding sites and formation of protein oligomers. Cooperative binding of the lambda Xis protein at the att DNA sites has been reported, and formation of this micronucleoprotein filament has been shown to drive DNA bending (26,27,30). Therefore, we speculate that the multiple Vef monomers bind at the att site and act similarly to bend the DNA, thereby driving the excision reaction.…”

“…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.…”

“…In TR integrase-encoding prophages, it was demonstrated that the excision event also requires an additional protein, an excisionase or recombination directionality factor (RDF) (24)(25)(26)(27)(28). In fact, it is believed that all TR integrases require an RDF to perform excision (20,29).…”

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

“…5). To investigate the DNA binding properties of mIHF, we chose dsDNA containing phage L5 attB (600 bp) or attP (546 bp) sites (24,56). Binding reactions were performed with 2 nM 32 P-labeled dsDNA having attP or attB site and increasing concentrations of mIHF.…”

Fluorescent Reporter DS6A Mycobacteriophages Reveal Unique Variations in Infectibility and Phage Production in Mycobacteria

“…5). First, the integrase gene (gp56) encodes a member of the tyrosine-integrase family of site-specific recombinases but has an unusual structure in which an excise-like domain is present within the gene, inserted approximately at codon 380; HHPred analysis shows significant similarity (E value, 10 Ϫ9 ) of this domain to the gp36 excise domain of mycobacteriophage Pukovnik (39). This is a highly unusual organization, and we are not aware of any other examples of this.…”