Two DOT1 enzymes cooperatively mediate efficient ubiquitin-independent histone H3 lysine 76 tri-methylation in kinetoplastids

ABSTRACT: In higher eukaryotes, a single DOT1 histone H3 lysine 79 (H3K79) methyltransferase processively produces H3K79me2/me3 from me0 through histone H2B mono-ubiquitin interaction. The early-branched kinetoplastid Trypanosoma brucei harbors the essential H3K76 di-methyltransferase DOT1A and the non-essential DOT1B H3K76 tri-methyltransferase, which methylate their substrates without H2B mono-ubiquitination. It is not known how these enzymes efficiently maintain H3K76me3 without ubiquitin interaction in vivo. Here we discovered distinct methylation kinetics of DOT1A and DOT1B in vitro and identified kinetoplastid-specific key residues by structural analysis of DOT1A. DOT1A contains a unique N-terminal ?-hairpin domain and a conserved C-terminal methyltransferase core domain that exhibits significant deviations in the methyltransferase motifs IV, VI, and X. The motif X sequence VYGE is substituted to CAKS in a non-conserved, flexible active-site loop, implying a distinct mechanism for substrate H3K76 recognition. Substitution of a basic to an acidic residue within the canonical motif VI (Gx6K) at the putative DOT1A-nucleosome interface is essential for DOT1A-nucleosome interaction, which would stabilize the enzyme-substrate complex without ubiquitin interactions. Mass spectrometry-based kinetic data demonstrate that DOT1A favors H3K76me0, while DOT1B favors the DOT1A products H3K76me1 and me2. These substrate preferences are determined by Ser and Ala residues within motif IV of DOT1A and DOT1B, respectively. We show in vitro that the distinct substrate preferences enable DOT1A and DOT1B to catalyze H3K76 tri-methylation eight times faster than DOT1B alone, suggesting that efficient ubiquitin-independent H3K76 tri-methylation can be achieved non-processively through the cooperative action of two DOT1 enzymes in vivo.

INSTRUMENT(S): Q Exactive Plus

ORGANISM(S): Trypanosoma Brucei (ncbitaxon:5691)

SUBMITTER: Benjamin A. Garcia  

PROVIDER: MSV000090680 | MassIVE | Wed Nov 09 10:40:00 GMT 2022

SECONDARY ACCESSION(S): PXD038070

REPOSITORIES: MassIVE