Yeast Spt16/Cdc68 and Pob3 form a heterodimer that acts in both DNA replication and transcription. This is supported by studies of new alleles of SPT16 described here. We show that Spt16-Pob3 enhances HO transcription through a mechanism that is affected by chromatin modification, since some of the defects caused by mutations can be suppressed by deleting the histone deacetylase Rpd3. While otherwise conserved among many eukaryotes, Pob3 lacks the HMG1 DNA-binding motif found in similar proteins such as the SSRP1 subunit of human FACT. SPT16 and POB3 display strong genetic interactions with NHP6A/B, which encodes an HMG1 motif, suggesting that these gene products function coordinately in vivo. While Spt16-Pob3 and Nhp6 do not appear to form stable heterotrimers, Nhp6 binds to nucleosomes and these Nhp6-nucleosomes can recruit Spt16-Pob3 to form SPN-nucleosomes. These complexes have altered electrophoretic mobility and a distinct pattern of enhanced sensitivity to DNase I. These results suggest that Spt16-Pob3 and Nhp6 cooperate to function as a novel nucleosome reorganizing factor.

FACT is a roughly 180 kDa heterodimeric protein complex important for managing the properties of chromatin in eukaryotic cells. Chromatin is a repressive barrier that plays an important role in protecting genomic DNA and regulating access to it. This barrier must be temporarily removed during transcription, replication, and repair, but it also must be rapidly restored to the original state afterwards. Further, the properties of chromatin are dynamic and must be adjusted as conditions dictate. FACT was identified as a factor that destabilizes nucleosomes in vitro, but it has now also been implicated as a central factor in the deposition of histones to form nucleosomes, as an exchange factor that swaps the histones within existing nucleosomes for variant forms, and as a tether that prevents histones from being displaced by the passage of RNA polymerases during transcription. FACT therefore plays central roles in building, maintaining, adjusting, and overcoming the chromatin barrier. This review summarizes recent results that have begun to reveal how FACT can promote what appear to be contradictory goals, using a simple set of binding activities to both enhance and diminish the stability of nucleosomes.

Summary The proteasome is an abundant protease that is critically important for numerous cellular pathways. Proteasomes are activated in vitro by three known classes of proteins/complexes, including Blm10/PA200. Here we report a 3.4Å resolution crystal structure of a proteasome-Blm10 complex, which reveals that Blm10 surrounds the proteasome entry pore in the 1.2 MDa complex to form a largely closed dome that is expected to restrict access of potential substrates. This architecture, and the observation that Blm10 induces a disordered proteasome gate structure, challenges the assumption that Blm10 functions as an activator of proteolysis in vivo. The Blm10 C-terminus binds in the same manner as seen for 11S activators and inferred for 19S/PAN activators, and indicates a unified model for gate opening. We also demonstrate that Blm10 acts to maintain mitochondrial function. Consistent with the structural data, the C-terminal residues of Blm10 are needed for this activity.

Summary FACT has been proposed to function by displacing an H2A–H2B dimer to form hexasomes. Results described here with yeast FACT (yFACT) suggest instead that nucleosomes are reorganized to a form with the original composition but a looser, more dynamic structure. First, yFACT enhances hydroxyl radical accessibility and endonuclease digestion in vitro at sites throughout the nucleosome, not just in regions contacted by H2A–H2B. Accessibility increases dramatically but the DNA remains partially protected. Second, increased nuclease sensitivity does not require displacement of dimers from the nucleosome. Third, yFACT is required for eviction of nucleosomes from the GAL1-10 promoter and the adjacent ORF in vivo, but most sites do not exhibit the preferential reduction in dimer occupancy expected for hexasome formation. We propose that yFACT promotes a reversible transition between two nucleosomal forms and that this is useful both to overcome the repressive chromatin barrier and to establish and maintain this barrier.

We have used DNA polymerase ␣ affinity chromatography to identify factors involved in eukaryotic DNA replication in the yeast Saccharomyces cerevisiae. Two proteins that bound to the catalytic subunit of DNA polymerase ␣ (Pol1 protein) are encoded by the essential genes CDC68/SPT16 and POB3. The binding of both proteins was enhanced when extracts lacking a previously characterized polymerase binding protein, Ctf4, were used. This finding suggests that Cdc68 and Pob3 may compete with Ctf4 for binding to Pol1. Pol1 and Pob3 were coimmunoprecipitated from whole-cell extracts with antiserum directed against Cdc68, and Pol1 was immunoprecipitated from whole-cell extracts with antiserum directed against the amino terminus of Pob3, suggesting that these proteins may form a complex in vivo. CDC68 also interacted genetically with POL1 and CTF4 mutations; the maximum permissive temperature of double mutants was lower than for any single mutant. Overexpression of Cdc68 in a pol1 mutant strain dramatically decreased cell viability, consistent with the formation or modulation of an essential complex by these proteins in vivo. A mutation in CDC68/SPT16 had previously been shown to cause pleiotropic effects on the regulation of transcription (J. The cdc68-1 mutation caused elevated rates of chromosome fragment loss, a phenotype characteristic of genes whose native products are required for normal DNA metabolism. However, this mutation did not affect the rate of loss or recombination for two intact chromosomes, nor did it affect the retention of a low-copy-number plasmid. The previously uncharacterized Pob3 sequence has significant amino acid sequence similarity with an HMG1-like protein from vertebrates. Based on these results and because Cdc68 has been implicated as a regulator of chromatin structure, we postulate that polymerase ␣ may interact with these proteins to gain access to its template or to origins of replication in vivo.ADNA replication is accomplished by large multiprotein complexes called replisomes, as demonstrated in studies using the replication machinery from Escherichia coli and its bacteriophages (39). The E. coli polymerase III holoenzyme is unusual in that it is stable enough to be purified intact, allowing its components to be identified (39). However, in most other systems the replisome is unstable, and components have been identified by using partial-function assays, genetic methods, in vitro complementation of extracts prepared from replicationdefective mutants, or affinity chromatography (39, 73). Studies of the replication machinery used by the eukaryotic virus simian virus 40 (SV40) showed that functional units in eukaryotic replisomes are remarkably similar to those found in prokaryotic systems (73). Although the proteins typically have little similarity at the amino acid level, the need for polymerases, processivity clamps, clamp loading proteins, helicases, primases, nucleases, and single-stranded DNA binding proteins appears to be universal (29,39,73). Some physical interactions have been detec...