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The N-terminus of Drosophila SU(VAR)3-9 mediates dimerization and regulates its methyltransferase activity.
- Source :
-
Biochemistry [Biochemistry] 2004 Mar 30; Vol. 43 (12), pp. 3740-9. - Publication Year :
- 2004
-
Abstract
- In most eukaryotes, the histone methyltransferase SU(VAR)3-9 and its orthologues play a major role in the function of centromeric heterochromatin. Although the methyltransferase domain is required for the formation of a fully functional centromere, mutations within other regions of the gene such as the N-terminus also have a strong impact on its in vivo function. To analyze the contribution of the N-terminus on the methyltransferase activity, we have expressed the full-length Drosophila SU(VAR)3-9 (dSU(VAR)3-9) together with various N-terminal deletions in Escherichia coli and analyzed the structural and enzymatic properties of the purified recombinant enzymes. Full-length dSU(VAR)3-9 specifically methylates lysine 9 within histone H3 on peptides, on intact histones, and, to a lesser extent, on nucleosomes. A detailed analysis of the reaction products shows that dSU(VAR)3-9 adds two methyl groups to an unmethylated H3 tail peptide in a nonprocessive manner. The full-length enzyme elutes with an apparent molecular weight of 160 kDa from a gel filtration column, which indicates the formation of a dimer. This property is dependent on an intact N-terminus. In contrast to the full-length enzymes, proteins lacking the N-terminus fail to dimerize, and show a 10-fold lower specific activity and a linear dependence of methyltransferase activity on enzyme concentration. A N-terminal peptide containing amino acids 1-152 of dSU(VAR)3-9 is sufficient to mediate this interaction in vitro. The dimerization of dSU(VAR)3-9 and the subsequent increase of its methyltransferase activity provide a starting point to understand the molecular details of the formation of heterochromatic structures in vivo.
- Subjects :
- Amino Acid Sequence
Animals
Catalysis
Dimerization
Drosophila Proteins genetics
Drosophila Proteins metabolism
Drosophila melanogaster genetics
Enzyme Activation genetics
Histone Methyltransferases
Histone-Lysine N-Methyltransferase genetics
Histone-Lysine N-Methyltransferase metabolism
Histones chemistry
Histones metabolism
Isoenzymes chemistry
Isoenzymes genetics
Isoenzymes metabolism
Kinetics
Lysine chemistry
Methylation
Methyltransferases genetics
Methyltransferases metabolism
Molecular Sequence Data
Peptide Fragments genetics
Peptide Fragments metabolism
Protein Binding genetics
Protein Methyltransferases
Protein Processing, Post-Translational genetics
Recombinant Proteins chemistry
Sequence Deletion genetics
Drosophila Proteins chemistry
Drosophila melanogaster enzymology
Histone-Lysine N-Methyltransferase chemistry
Methyltransferases chemistry
Peptide Fragments chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 43
- Issue :
- 12
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15035645
- Full Text :
- https://doi.org/10.1021/bi035964s