SETD3 is an actin histidine-N 3 methyltransferase, whereas other characterized SET-domain enzymes are protein lysine methyltransferases. We report that in a pre-reactive complex SETD3 binds the N 3 -protonated form (N 3 -H) of actin His73, and in a post-reactive product complex, SETD3 generates the methylated histidine in an N 1 -protonated (N 1 -H) and N 3 -methylated form. During the reaction, the imidazole ring of His73 rotates ~105°, which shifts the proton from N 3 to N 1 , thus ensuring that the target atom N 3 is deprotonated prior to the methyl transfer. Under the conditions optimized for lysine deprotonation, SETD3 has weak lysine methylation activity on an actin peptide in which the target His73 is substituted by a lysine. The structure of SETD3 with Lys73-containing peptide reveals a bent conformation of Lys73, with its side chain aliphatic carbons tracing along the edge of imidazole ring and the terminal ε-amino group occupying a position nearly identical to the N 3 atom of unmethylated histidine.

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Caulobacter crescentus relies on DNA methylation by the cell cycle-regulated methyltransferase (CcrM) in addition to key transcription factors to control the cell cycle and direct cellular differentiation. CcrM is shown here to efficiently methylate its cognate recognition site 5'-GANTC-3' in single-stranded and hemimethylated double-stranded DNA. We report the K, k, k, and K for single-stranded and hemimethylated substrates, revealing discrimination of 10-fold for noncognate sequences. The enzyme also shows a similar discrimination against single-stranded RNA. Two independent assays clearly show that CcrM is highly processive with single-stranded and hemimethylated DNA. Collectively, the data provide evidence that CcrM and other DNA-modifying enzymes may use a new mechanism to recognize DNA in a key epigenetic process.

The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. Here we present the structure of CcrM in complex with double-stranded DNA containing the recognition sequence. CcrM contains an N-terminal methyltransferase domain and a C-terminal nonspecific DNA-binding domain. CcrM is a dimer, with each monomer contacting primarily one DNA strand: the methyltransferase domain of one molecule binds the target strand, recognizes the target sequence, and catalyzes methyl transfer, while the C-terminal domain of the second molecule binds the non-target strand. The DNA contacts at the 5-base pair recognition site results in dramatic DNA distortions including bending, unwinding and base flipping. The two DNA strands are pulled apart, creating a bubble comprising four recognized base pairs. The five bases of the target strand are recognized meticulously by stacking contacts, van der Waals interactions and specific Watson–Crick polar hydrogen bonds to ensure high enzymatic specificity.

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