O -Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNASep binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep218, Sep222). This system has general utility in protein engineering, molecular biology, and disease research.

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Two cytotoxic proteins, bovine pancreatic ribonuclease A (RNase A), and a restriction endonuclease from H a e n q~h i l u s parainfluenzae ( H p a l ) , were produced using a novel semisynthetic approach that utilizes a protein splicing element, an intein, to generate a reactive thioester at the C-terminus of a recombinant protein. Nucleophilic attack on this thioester by the N-terminal cysteine of a synthetic peptide ultimately leads to the ligation of the two reactants through a native peptide bond. This strategy was used to produce RNase A and HpaI by isolating inactive truncated forms of these proteins, the first 109 and 223 amino acids of RNase A and HpaI, respectively, as fusion proteins consisting of the target protein, an intein, and a chitin binding domain. Thiol-induced cleavage of the precursor led to the liberation of the target protein with a C-terminal thioester-tag. Addition of synthetic peptides representing the amino acids missing from the truncated forms led to the generation of full-length products that displayed catalytic activity indicative of the wild-type enzymes. The turnover numbers and K,,, for ligated and renatured RNase A were 8.2 s -' and 1.5 mM, in good agreement with reported values of 8.3 s -' and 1.2 mM (Hodges & Merrifield, 1975). Ligated Hpal had a specific activity of 0.5-1.5 X 10' U/mg, which compared favorably with the expected value of 1-2 X 10' U/mg (J. Benner, unpubl. obs.). Besides assisting in the production of cytotoxic proteins, this technique could allow the easy insertion of unnatural amino acids into a protein sequence.

Methylation of small molecules and macromolecules is crucial in metabolism, cell signaling, and epigenetic programming and is most often achieved by S-adenosylmethionine (SAM)-dependent methyltransferases. Most employ an S(N)2 mechanism to methylate nucleophilic sites on their substrates, but recently, radical SAM enzymes have been identified that methylate carbon atoms that are not inherently nucleophilic via the intermediacy of a 5'-deoxyadenosyl 5'-radical. We have determined the mechanisms of two such reactions targeting the sp(2)-hybridized carbons at positions 2 and 8 of adenosine 2503 in 23S ribosomal RNA, catalyzed by RlmN and Cfr, respectively. In neither case is a methyl group transferred directly from SAM to the RNA; rather, both reactions proceed by a ping-pong mechanism involving intermediate methylation of a conserved cysteine residue.

Inheritance of epigenetic information encoded by cytosine DNA methylation patterns is crucial for mammalian cell survival, in large part through the activity of the maintenance DNA methyltransferase (DNMT1). Here, we show that SET7, a known histone methyltransferase, is involved in the regulation of protein stability of DNMT1. SET7 colocalizes and directly interacts with DNMT1 and specifically monomethylates Lys-142 of DNMT1. Methylated DNMT1 peaks during the S and G 2 phases of the cell cycle and is prone to proteasome-mediated degradation. Overexpression of SET7 leads to decreased DNMT1 levels, and siRNA-mediated knockdown of SET7 stabilizes DNMT1. These results demonstrate that signaling through SET7 represents a means of DNMT1 enzyme turnover.DNA methyltransferase ͉ methylated lysine ͉ proteasome ͉ protein degradation M ammalian DNA methylation is essential for development and is controlled by a variety of factors including 3 active DNA cytosine methyltransferases (DNMT1, DNMT3A, and DNMT3B) and a methyltransferase-like protein, DNMT3L (1-4). DNMT1 encodes the maintenance DNA methyltransferase (DNMT) responsible for methylating hemimethylated CpG sites shortly after DNA replication, and it is assisted by an accessory factor capable of recognizing hemimethylated DNA called UHRF1 (5, 6). Aberrant DNA methylation of CpG islandcontaining promoters leads to permanent silencing of genes in both physiological and pathological contexts and specifically in cancer cells (7). In cancer cells, disruption of DNMT1 resulted in hemimethylation of a fifth of the CpG sites in the genome and activation of the G 2 /M checkpoint, leading to arrest in the G 2 phase of the cell cycle (8). Apart from DNA methylationmediated gene silencing, DNMT1 also binds to several transcriptional inhibitors and represses gene expression in a DNA methylation-independent manner (9-11). Pharmacological inhibitors of DNMT1 [5-azacytidine (5-aza-CR) and its deoxy analog, 5-aza-2Ј-deoxycytidine (5-aza-CdR)] get incorporated into newly-synthesized DNA (12, 13). Once incorporated into DNA, these compounds form covalent complexes with DNMTs, thereby depleting active enzymes (14, 15) and activating gene expression (16). Recently, 5-aza-CdR-induced depletion of DNMT1 was shown to be mediated by proteasomal pathways in mammalian nuclei (17). However, little is known about other factors regulating DNMT1 levels in cells. Here, we show that DNMT1 stability is regulated by protein methylation coupled to proteasome-mediated protein degradation through the protein methyltransferase activity of SET7. Results DNMT1 Colocalizes and Associates with and Is Methylated by SET7.We used gel filtration and Western blot analysis to analyze DNMT1 from nuclear extracts. Using a highly-specific antibody we observed a major species of DNMT1 at 185 kDa and a higher molecular mass minor species (Fig. 1A). It seemed likely that this minor species of DNMT1 may be posttranslationally modified (18). To test whether DNMT1 might be modified by protein methylation, recombinant DNMT1 wa...