yFACT (heterodimers of Saccharomyces cerevisiae Spt16-Pob3 combined with Nhp6) binds to and alters the properties of nucleosomes. The essential function of yFACT is not disrupted by deletion of the N-terminal domain (NTD) of Spt16 or by mutation of the middle domain of Pob3, but either alteration makes yeast cells sensitive to DNA replication stress. We have determined the structure of the Spt16 NTD and find evidence for a conserved potential peptide-binding site. Pob3-M also contains a putative binding site, and we show that these two sites perform an overlapping essential function. We find that yFACT can bind the N-terminal tails of some histones and that this interaction is important for yFACT-nucleosome binding. However, neither the Spt16 NTD nor a key residue in the putative Pob3-M-binding site was required for interactions with histone N termini or for yFACT-mediated nucleosome reorganization in vitro. Instead, both potential binding sites interact functionally with the C-terminal docking domain of the histone H2A. yFACT therefore appears to make multiple contacts with different sites within nucleosomes, and these interactions are partially redundant with one another. The docking domain of H2A is identified as an important participant in maintaining stability during yFACT-mediated nucleosome reorganization, suggesting new models for the mechanism of this activity.yFACT (yeast facilitator of chromatin transcription or transactions) is a heterodimer of the Saccharomyces cerevisiae Spt16 and Pob3 proteins that is assisted in vivo and in vitro by the high mobility group type B domain DNA-binding protein Nhp6 (1, 2). In vitro, yFACT binds to histones (3, 4) and can alter the accessibility of DNA within nucleosomes without hydrolyzing ATP and without repositioning the histone octamer core relative to the DNA (5-7). This activity is different from ATP-dependent chromatin remodeling and has been called nucleosome reorganization (6). yFACT and related FACT complexes from other eukaryotes are needed for both normal regulation of transcription (5, 8 -11) and for DNA replication (12-20). Reorganization activity therefore appears to be important in a range of chromatin-based processes, including initiation and elongation of transcription, establishment and maintenance of normal chromatin, and survival during DNA replication stress. Consistent with this broad functional importance, FACT family members have been found in all eukaryotes examined, and at least one of the subunits is essential for viability in all cases reported (9,21,19,22).FACT complexes contain several distinct structural domains (16, 23), but little is known about how these domains contribute to FACT function. The middle domain of Pob3 (Pob3-M) forms two pleckstrin homology (PH) 4 folds that are closely juxtaposed (23), with highly conserved surface residues forming a patch in a region often associated with binding sites in PH domain proteins (23). Altering this patch caused increased sensitivity to hydroxyurea (HU) (23), a toxin that blocks dNTP synt...

FACT (FAcilitates Chromatin Transcription/Transactions) plays a central role in transcription and replication in eukaryotes by both establishing and overcoming the repressive properties of chromatin. FACT promotes these opposing goals by interconverting nucleosomes between the canonical form and a more open reorganized form. In the forward direction, reorganization destabilizes nucleosomes, while the reverse reaction promotes nucleosome assembly. Nucleosome destabilization involves disrupting contacts among histone H2A-H2B dimers, (H3-H4) 2 tetramers, and DNA. Here we show that mutations that weaken the dimer:tetramer interface in nucleosomes suppress defects caused by FACT deficiency in vivo in the yeast Saccharomyces cerevisiae. Mutating the gene that encodes the Spt16 subunit of FACT causes phenotypes associated with defects in transcription and replication, and we identify histone mutants that selectively suppress those associated with replication. Analysis of purified components suggests that the defective version of FACT is unable to maintain the reorganized nucleosome state efficiently, whereas nucleosomes with mutant histones are reorganized more easily than normal. The genetic suppression observed when the FACT defect is combined with the histone defect therefore reveals the importance of the dynamic reorganization of contacts within nucleosomes to the function of FACT in vivo, especially to FACT's apparent role in promoting progression of DNA replication complexes. We also show that an H2B mutation causes different phenotypes, depending on which of the two similar genes that encode this protein are altered, revealing unexpected functional differences between these duplicated genes and calling into question the practice of examining the effects of histone mutants by expressing them from a single plasmid-borne allele.