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Pob3 is the yeast functional homolog of metazoan SSRP1 and a core subunit of the
histone chaperone complex FACT, built around an essential Spt16-Pob3 heterodimer
that functions together with HMGB proteins (notably Nhp6) to engage nucleosomes.
"In budding yeast, FACT is built around an essential Spt16–Pob3 heterodimer and functions together with HMGB proteins (notably Nhp6) to engage nucleosomes during chromatin-based processes."
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FACT performs ATP-independent nucleosome reorganization and has no intrinsic
ATPase activity, consistent with Pob3 being a structural histone-handling
module rather than an enzyme catalyzing a chemical transformation.
"FACT (FAcilitates Chromatin Transcription/Transactions) is a conserved, abundant histone chaperone complex implicated in transcription, DNA replication, and DNA repair through ATP-independent nucleosome reorganization (i.e., without an intrinsic ATPase)."
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Pob3 is a multi-domain protein: an N-terminal domain (~aa 1-220) mediating
dimerization with Spt16, a middle domain (~aa 237-447), and a C-terminal
intrinsically disordered acidic domain (CTD) that is a major in vivo
histone-binding module.
"High-resolution in vivo interaction mapping and domain analyses describe Pob3 as a multi-domain protein with (i) an N-terminal domain (NTD; ~aa 1–220) involved in complex formation/dimerization with Spt16; (ii) a middle domain (MD; ~aa 237–447); and (iii) a C-terminal intrinsically disordered acidic domain (CTD), which is a major histone-binding module in vivo."
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The acidic Pob3 CTD provides direct H2A-H2B histone-binding capacity; in vivo
photo-crosslinking maps multiple CTD sites that directly contact H2A/H2B, and
the CTD binds H2A-H2B with low micromolar affinity in vitro.
"A central mechanistic role for Pob3 is to provide direct histone-binding capacity—especially to H2A–H2B—through its acidic CTD. In vivo photo-crosslinking identifies multiple Pob3-CTD sites that directly contact H2A/H2B"
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A monopartite C-terminal NLS (residues 544-552, RPSKKPKVE) directs Pob3
nuclear import; importin-alpha binding to this NLS is negatively coupled to
Pob3-H2A/H2B binding (importin-alpha reduces Pob3-H2A/H2B crosslinking ~2-fold),
coupling trafficking to histone-chaperone engagement.
"A monopartite C-terminal NLS was mapped to Pob3 residues 544–552 (sequence **RPSKKPKVE**, NLS score **9.5/10**). Deleting this segment (Δ544–552) or mutating key residues (e.g., K547M) disrupts nuclear localization of Pob3-GFP fusions."
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FACT engagement with nucleosomes is transcription-dependent: FACT-bound
nucleosomes show altered MNase protection, and transcription inhibition
restores MNase resistance, indicating preferential engagement with
RNAPII-disrupted nucleosomes.
"In vivo, FACT’s interaction with nucleosomes is transcription-dependent, and FACT-bound nucleosomes show altered MNase protection patterns versus bulk chromatin; transcription inhibition restores MNase resistance, supporting preferential engagement with RNAP-disrupted nucleosomes."
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Recruitment of FACT to gene coding regions depends on the histone H3 tail in
an acetylation-dependent manner; loss of the H3 acetyltransferase Gcn5 or H3
tail lysine mutation reduces FACT occupancy.
"Recruitment of FACT to coding sequences depends on the histone H3 tail in an acetylation-dependent manner; deletion of the H3 acetyltransferase Gcn5 or mutation of H3 tail lysines reduces FACT occupancy at tested genes"
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Mutations affecting FACT subunits cause cryptic transcription initiation
within gene bodies and altered chromatin states, consistent with a role for
Pob3/FACT in restoring nucleosome organization during and after transcription.
"Mutations affecting FACT subunits are associated with cryptic transcription initiation within gene bodies and altered chromatin states, consistent with a key role for FACT/Pob3 in restoring or maintaining nucleosome organization during and after transcription."
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The Pob3 acidic CTD contributes to replication-linked functions: CTD deletion
yields hydroxyurea sensitivity, supporting a role in DNA replication and the
replication stress response.
"The Pob3 acidic CTD contributes to replication-linked functions: CTD deletion (ΔS491–E543) yields hydroxyurea sensitivity, supporting a role in DNA replication/replication stress responses."
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A 2024 preprint proposes that FACT forms an H3-H4-mediated bridge to DNA
polymerase alpha via Pol1's N-terminal domain, with the ternary complex
stimulating Pol alpha activity to coordinate lagging-strand synthesis with
nucleosome assembly (S-phase-specific interactions in yeast).
"A recent mechanistic proposal (preprint) suggests that FACT can form an H3–H4–mediated bridge to DNA polymerase α (Pol α) via Pol1’s N-terminal domain: H3–H4 promotes FACT–Pol α association, and the resulting ternary complex stimulates Pol α polymerase activity to coordinate lagging-strand synthesis with nucleosome assembly."
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Genome-wide mapping shows transcription-dependent enrichment of FACT (Spt16
measured; Pob3 as obligate partner) near the 3' ends of all Pol III-transcribed
genes, influencing downstream nucleosome organization.
"FACT enrichment reported at **3′ ends of all Pol III-transcribed genes**"
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Across modalities, Pob3 is a nuclear, chromatin-bound histone chaperone
subunit whose acidic intrinsically disordered CTD provides direct H2A-H2B
engagement and whose cooperation with Spt16 and Nhp6 lets FACT recognize and
stabilize transcription- or replication-perturbed nucleosomes; it is not an
enzyme but a structural histone-handling module.
"Pob3 is not an enzyme catalyzing a chemical transformation, but rather a **structural histone-handling module** that helps maintain chromatin integrity during high-flux DNA transactions."