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Dot1p is the sole histone H3 lysine-79 (H3K79) methyltransferase in budding
yeast, a non-SET SAM-dependent enzyme that methylates H3K79 on
nucleosomes/chromatin (not free histones), producing mono-, di-, and
trimethylation states via a distributive (non-processive) mechanism.
"Dot1p is a non-SET, SAM-dependent methyltransferase that methylates H3K79 specifically on nucleosomes/chromatin (not free histones), producing mono-, di-, and tri-methylation states via a distributive (non-processive) mechanism."
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The physiologically relevant substrate is nucleosomal H3K79; Dot1p does not
efficiently methylate free histone H3 or free histones, requiring the
nucleosomal context for activity.
"A central defining feature is that Dot1p is **nucleosome/chromatin dependent** and does **not** efficiently methylate free histone H3/free histones; thus, the physiologically relevant substrate is nucleosomal H3K79 within chromatin."
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Dot1 activity is allosterically stimulated by H2B-K123 monoubiquitination
(H2Bub1) and by H4K16 acetylation (H4K16ac), which primarily increase
catalytic turnover (kcat) rather than nucleosome binding affinity, boosting
di-/tri-methylation capacity.
"- **H2B-K123 monoubiquitination (H2Bub1)** stimulates Dot1-dependent H3K79 methylation, especially higher methylation states (di-/tri-methylation). (wood2018dot1landh3k79 pages 1-3, farooq2016themanyfaces pages 3-4)
- **H4K16 acetylation (H4K16ac)** directly and allosterically stimulates Dot1 catalysis and cooperates with H2Bub1."
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Quantitative kinetics show stimulation is catalytic, not binding-driven:
unmodified nucleosomes Km = 381 nM, kcat = 0.85 min-1; H4K16ac kcat = 17.5
min-1; H2Bub kcat = 9.71 min-1; doubly modified kcat = 35.5 min-1, with Kd
essentially unchanged (~70-83 nM).
"On unmodified nucleosomes, Km = 381 ± 227 nM and kcat = 0.85 ± 0.21 min⁻¹; on H2Bub nucleosomes, Km = 387 ± 59.2 nM and kcat = 9.71 ± 0.54 min⁻¹."
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Dot1/H3K79 methylation antagonizes Sir complex spreading at silencing
boundaries; H3K79 methylation blocks Sir3 binding, while Dot1 loss or H3K79
mutation impairs telomeric silencing by mislocalizing Sir proteins.
"Dot1p was originally identified through effects on telomeric silencing, and mechanistically H3K79 methylation antagonizes Sir3 binding and Sir complex localization/spreading. Dot1 deletion or H3K79 mutation compromises telomeric silencing and alters Sir protein localization"
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Dot1p is nuclear and chromatin-associated, acting on nucleosomal H3K79
across euchromatic transcribed regions (gene bodies); H3K79 methylation is
reported across ~90% of the yeast genome.
"Dot1p function is inherently chromatin-associated because it methylates nucleosomal H3K79 and is enriched across transcribed regions (gene bodies) in euchromatin."
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Dot1/H3K79 methylation contributes to genome stability and DNA damage
checkpoint signaling; Dot1 or H3K79 defects impair recruitment of DNA damage
response factors and cause G1 and intra-S checkpoint defects.
"Dot1/H3K79 methylation contributes to genome stability and DNA damage checkpoint signaling. Defects in Dot1/H3K79 methylation can impair recruitment of DNA damage response factors and checkpoint activation (e.g., G1 and intra-S checkpoint defects described in yeast contexts)"
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Beyond catalysis, Dot1p has intrinsic, methylation-independent histone
chaperone activity that regulates nucleosome dynamics and histone exchange;
it can assemble core histones into nucleosomes, and a nucleosome-binding
domain (residues 101-140) is required, with catalytically inactive mutants
still able to stimulate remodeling.
"A major yeast-specific advance is evidence that Dot1p has intrinsic **histone chaperone activity** that regulates nucleosome dynamics and histone exchange independently of methyltransferase activity:
- Dot1p can assemble core histones into nucleosomes and facilitate ATP-dependent chromatin remodeling in vitro.
- A Dot1 nucleosome-binding domain (residues 101–140) is required for chaperone/remodeling stimulation.
- Catalytically inactive Dot1 mutants can still stimulate remodeling, supporting methylation-independent function."
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Dot1 can reciprocally promote H2B-K123 ubiquitination independent of its
methyltransferase activity (including in catalytically dead variants),
dependent on the H2B-K123 site and the Bre1 ligase.
"In addition to being regulated by H2Bub1, Dot1 overexpression was reported to increase H2Bub1 levels through a mechanism independent of Dot1 catalytic activity (including catalytically dead Dot1 variants), with dependence on the H2B-K123 site and on the Bre1 ligase"