The Saccharomyces cerevisiae DASH complex is an essential microtubule-binding component of the kinetochore. We coexpressed all ten subunits of this assembly in Escherichia coli and purified a single complex, a approximately 210-kDa heterodecamer with an apparent stoichiometry of one copy of each subunit. The hydrodynamic properties of the recombinant assembly are indistinguishable from those of the native complex in yeast extracts. The structure of DASH alone and bound to microtubules was visualized by EM. The free heterodecamer is relatively globular. In the presence of microtubules, DASH oligomerizes to form rings and paired helices that encircle the microtubules. We discuss potential roles for such collar-like structures in maintaining microtubule attachment and spindle integrity during chromosome segregation.

The yeast DASH complex is a heterodecameric component of the kinetochore necessary for accurate chromosome segregation. DASH forms closed rings around microtubules with a large gap between the DASH ring and the microtubule cylinder. We characterized the microtubule-binding properties of limited proteolysis products and subcomplexes of DASH, thus identifying candidate polypeptide extensions involved in establishing the DASH-microtubule interface. The acidic C-terminal extensions of tubulin subunits are not essential for DASH binding. We also measured the molecular mass of DASH rings on microtubules with scanning transmission electron microscopy and found that approximately 25 DASH heterodecamers assemble to form each ring. Dynamic association and relocation of multiple flexible appendages of DASH may allow the kinetochore to translate along the microtubule surface.

A protein model was developed for studying the interaction between cysteine residues and the helix dipole. Site-directed mutagenesis was used to introduce cysteine residues at the N-terminus of helix H in recombinant sperm whale myoglobin. Based on the difference in thiol pK a between folded proteins and an unfolded peptide, the energy of interaction between the thiolate and the helix dipole was determined. Thiolates at the N1 and N2 positions of the helix were stabilized by 0.3 kcal/mole and 0.7 kcal/mole, respectively. A thiolate at the Ncap position was stabilized by 2.8 kcal/mole, and may involve a hydrogen bond. In context with other studies, an experimentally observed helix dipole effect may be defined in terms of two distinct components. A charge-dipole component involves electrostatic interactions with peptide bond dipoles in the first two turns of the helix and affects residues at all positions of the terminus; a hydrogen bond component involves one or more backbone amide groups and is only possible at the capping position due to conformational restraints elsewhere. The nature and magnitude of the helix dipole effect is, therefore, position-dependent. Results from this model system were used to interpret cysteine reactivity in rodent hemoglobins and the thioredoxin family.

The human CCCTC-binding factor, CTCF, regulates transcription of the double-stranded DNA genomes of herpesviruses. The architectural complex cohesin and RNA Polymerase II also contribute to this organization. We profiled the occupancy of CTCF, cohesin, and RNA Polymerase on the episomal genome of the Epstein-Barr virus in a cell culture model of latent infection. CTCF colocalizes with cohesin but not RNA Polymerase. CTCF and cohesin bind specific sequences throughout the genome that are found not just proximal to the regulatory elements of latent genes, but also near lytic genes. In addition to tracking with known transcripts, RNA Polymerase appears at two unannotated positions, one of which lies within the latent origin of replication. The widespread occupancy profile of each protein reveals binding near or at a myriad of regulatory elements and suggests context-dependent functions.

The human genome is structurally organized in three-dimensional space to facilitate functional partitioning of transcription. We learned that the latent episome of the human Epstein-Barr virus (EBV) preferentially associates with gene-poor chromosomes and avoids gene-rich chromosomes. Kaposi's sarcoma-associated herpesvirus behaves similarly, but human papillomavirus does not. Contacts on the EBV side localize to OriP, the latent origin of replication. This genetic element and the EBNA1 protein that binds there are sufficient to reconstitute chromosome association preferences of the entire episome. Contacts on the human side localize to gene-poor and AT-rich regions of chromatin distant from transcription start sites. Upon reactivation from latency, however, the episome moves away from repressive heterochromatin and toward active euchromatin. Our work adds three-dimensional relocalization to the molecular events that occur during reactivation. Involvement of myriad interchromosomal associations also suggests a role for this type of long-range association in gene regulation.IMPORTANCE The human genome is structurally organized in three-dimensional space, and this structure functionally affects transcriptional activity. We set out to investigate whether a double-stranded DNA virus, Epstein-Barr virus (EBV), uses mechanisms similar to those of the human genome to regulate transcription. We found that the EBV genome associates with repressive compartments of the nucleus during latency and with active compartments during reactivation. This study advances our knowledge of the EBV life cycle, adding three-dimensional relocalization as a novel component to the molecular events that occur during reactivation. Furthermore, the data add to our understanding of nuclear compartments, showing that disperse interchromosomal interactions may be important for regulating transcription.