Nuclear egress, the trafficking of herpesvirus nucleocapsids from the nucleus to the cytoplasm, involves two conserved viral proteins that form a complex at the nuclear envelope, referred to as the nuclear egress complex. In human cytomegalovirus, these two proteins are called UL50 and UL53. To study UL50 and UL53 in molecular detail, these proteins were expressed in bacteria and purified. To obtain highly expressed, pure proteins, it was necessary to truncate both constructs based on sequence conservation and predicted secondary structural elements. Size exclusion chromatography and analytical ultracentrifugation studies indicated that the truncated form of UL50 is a monomer in solution, that the truncated form of UL53 is a homodimer, and that, when mixed, the two proteins form a heterodimer. To identify residues of UL53 crucial for homodimerization and for heterodimerization with UL50, we constructed and expressed mutant forms of UL53 containing alanine substitutions in a predicted helix. Isothermal titration calorimetry was used to measure the binding affinities of the UL53 mutants to UL50. UL53 residues, the replacement of which reduced binding to UL50, form a surface on one face of the predicted helix. Moreover, most of the substitutions that reduce UL53-UL50 interactions also reduced homodimerization. Substitutions that reduced the interaction between UL50 and UL53 in vitro also reduced colocalization of full-length UL50 and UL53 at the nuclear rim in transfected cells. These results demonstrate direct protein-protein interactions between these proteins that are likely to be mediated by a helix, and they have implications for understanding nuclear egress and for drug discovery.The delivery of newly assembled herpes capsids from the nucleus to the cytoplasm is a process known as nuclear egress (12). This process is still rather poorly understood. Although it is generally recognized that early steps of nuclear egress entail nucleocapsids' gaining access to the inner nuclear membrane, followed by primary envelopment of the nucleocapsids and budding through the inner nuclear membrane, there are currently three models for the remaining steps (13,14). Even the early steps in nuclear egress are not understood mechanistically although the formation of a specific nuclear egress complex (NEC) (10,11,19,21) has been shown to be crucial. In alphaherpesviruses such as herpes simplex virus (HSV) and pseudorabies virus (PRV), the NEC consists of two proteins called UL34 and UL31 (11). Throughout the alpha, beta, and gamma subfamilies of herpesviruses, highly conserved homologs of these two proteins are found to form an NEC at the nuclear rim in all studied cases (1,4,5,8,10,(17)(18)(19)25). Remarkably, in transfected cells, the PRV NEC, without any other viral proteins, is capable of remodeling the nuclear membrane to form vesicles that resemble the primary enveloped particles observed during productive infection (7).The human cytomegalovirus (HCMV) NEC is composed of UL50 and UL53. UL50 of HCMV was predicted to have a p...

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 rate constant for the phosphoryl transfer step in site-specific DNA cleavage by EcoRV endonuclease has been determined as a function of pH and identity of the required divalent metal ion cofactor, for both wild-type and T93A mutant enzymes. These measurements show bell-shaped pH-rate curves for each enzyme in the presence of Mg2+ as a cofactor, indicating general base catalysis for the nucleophilic attack of hydroxide ion on the scissile phosphate, and general acid catalysis for protonation of the leaving 3'-O anion. The kinetic data support a model for phosphoryl transfer based on wild-type and T93A cocrystal structures, in which the ionizations of two distinct metal-ligated waters respectively generate the attacking hydroxide ion and the proton for donation to the leaving group. The model concurs with recent observations of two metal ions bound in the active sites of the type II restriction endonucleases BamHI and BglI, suggesting the possibility of a similar catalytic mechanism functioning in many or all members of this enzyme family.