Abstract
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.
Highlights
The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication
Like the mammalian DNA cytosine methyltransferase (MTase) Dnmt[12], which is essential for maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks[3,4], the DNA adenine MTase (Dam) in Escherichia coli and cell cycle-regulated DNA MTase (CcrM) in Caulobacter crescentus are responsible for maintenance methylation of GATC or GANTC immediately after replication, respectively[5,6]
The DNA-protein contacts are concentrated on the five base pairs of the recognition site by a CcrM dimer (Fig. 1a, b)
Summary
The Caulobacter crescentus cell cycle-regulated DNA methyltransferase (CcrM) methylates the adenine of hemimethylated GANTC after replication. 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. Many sequence-specific DNA binding proteins exercise their effects by binding to specific genomic regions Nucleotide modifying enzymes, such as DNA MTases that methylate the target nucleotide (cytosine or adenine) within a specific DNA sequence, face the obstacle of having to bring the intrahelical target nucleotide into a concave catalytic pocket. By means of X-ray crystallography, we show that a CcrM dimer binds DNA by opening up double stranded DNA at the recognition site, a DNA recognition mechanism that likely contributes to the enzyme’s sequence discrimination
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