Abstract
In S. cerevisiae, the lysine methyltransferase Set1 is a member of the multiprotein complex COMPASS. Set1 catalyzes mono-, di- and trimethylation of the fourth residue, lysine 4, of histone H3 using methyl groups from S-adenosylmethionine, and requires a subset of COMPASS proteins for this activity. The methylation activity of COMPASS regulates gene expression and chromosome segregation in vivo. To improve understanding of the catalytic mechanism of Set1, single amino acid substitutions were made within the SET domain. These Set1 mutants were evaluated in vivo by determining the levels of K4-methylated H3, assaying the strength of gene silencing at the rDNA and using a genetic assessment of kinetochore function as a proxy for defects in Dam1 methylation. The findings indicate that no single conserved active site base is required for H3K4 methylation by Set1. Instead, our data suggest that a number of aromatic residues in the SET domain contribute to the formation of an active site that facilitates substrate binding and dictates product specificity. Further, the results suggest that the attributes of Set1 required for trimethylation of histone H3 are those required for Pol II gene silencing at the rDNA and kinetochore function.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Amino Acid Sequence
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Amino Acid Substitution
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Catalytic Domain
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism
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Conserved Sequence
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DNA, Ribosomal / genetics
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DNA, Ribosomal / metabolism
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Gene Expression Regulation, Fungal*
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Gene Silencing
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Histone-Lysine N-Methyltransferase / antagonists & inhibitors
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Histone-Lysine N-Methyltransferase / genetics
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Histone-Lysine N-Methyltransferase / metabolism*
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Histones / genetics
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Histones / metabolism*
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Kinetochores / pathology
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Lysine / metabolism
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Microtubule-Associated Proteins / genetics
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Microtubule-Associated Proteins / metabolism
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Molecular Sequence Data
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Mutation
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RNA, Small Interfering / genetics
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S-Adenosylmethionine / metabolism
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae Proteins / antagonists & inhibitors
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Sequence Alignment
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Substrate Specificity
Substances
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Cell Cycle Proteins
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DAM1 protein, S cerevisiae
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DNA, Ribosomal
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Histones
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Microtubule-Associated Proteins
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RNA, Small Interfering
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Saccharomyces cerevisiae Proteins
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S-Adenosylmethionine
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Histone-Lysine N-Methyltransferase
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SET1 protein, S cerevisiae
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Lysine