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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Summary A large group of E3 ubiquitin ligases is formed by the multisubunit SCF complex, whose core complex (Rbx1/Cul1-Cdc53/Skp1) binds one of many substrate recruiting F-box proteins to form an array of SCF ligases with diverse substrate specificities. It has long been thought that ubiquitylation by SCF ligases is regulated at the level of substrate binding. Here we describe an alternative mechanism of SCF regulation by active dissociation of the F-box subunit. We show that cadmium stress induces selective recruitment of the AAA+ ATPase Cdc48/p97 to catalyze dissociation of the F-box subunit from the yeast SCFMet30 ligase to block substrate ubiquitylation and trigger downstream events. Our results not only provide an additional layer of ubiquitin ligase regulation but also suggest that targeted, signal-dependent dissociation of multisubunit enzyme complexes is an important mechanism in control of enzyme function.

SUMMARYThe phage-encoded Xis protein is the major determinant controlling the direction of recombination in phage lambda. Xis is a winged-helix DNA binding protein that cooperatively binds to the attR recombination site to generate a curved microfilament, which promotes assembly of the excisive intasome but inhibits formation of an integrative intasome. We find that lambda synthesizes surprisingly high levels of Xis immediately upon prophage induction when excision rates are maximal. However, because of its low sequence-specific binding activity, exemplified by a 1.9 Å co-crystal structure of a nonspecifically bound DNA complex, Xis is relatively ineffective at promoting excision in vivo in the absence of the host Fis protein. Fis binds to a segment in attR that almost entirely overlaps one of the Xis binding sites. Instead of sterically excluding Xis binding from this site, as has been previously believed, we show that Fis enhances binding of all three Xis protomers to generate the microfilament. A specific Fis-Xis interface is supported by the effects of mutations within each protein, and relaxed, but not completely sequence-neutral, binding by the central Xis protomer is supported by the effects of DNA mutations. We present a structural model for the 50 bp curved Fis-Xis cooperative complex that is assembled between the arm and Holliday junction Int binding sites whose trajectory places constraints on models for the excisive intasome structure.

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