The cell cycle-regulated DNA adenine methyltransferase CcrM opens a bubble at its DNA recognition site

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.

Cell cycle regulated DNA methyltransferase: fluorescent tracking of a DNA strand-separation mechanism and identification of the responsible protein motif

“…As a control, no fluorescence changes are observed with M.HhaII, which recognizes the same site (GANTC), lacks the C-terminal segment, and which shows no activity with single-stranded DNA ( 13 ). No CcrM-dependent changes in fluorescence are observed if Pyrrolo-dC is positioned outside the recognition site (Figure 2 , P2 NT , P3 T , P8 T ) which is consistent with the cocrystal structure ( 17 ). Placement of Pyrrolo-dC at the C position within the recognition site in the target and non-target strands (Figure 2 , P6 NT , P7 T ) also shows no to little change in fluorescence upon CcrM binding.…”

“…The CcrM-DNA cocrystal structure reveals that four of the five base pairs within the recognition site are disrupted upon CcrM binding, with the target strand positioned away from the complementary non-target strand (Figure 1 ) ( 17 ). Our interest was to provide a means to track the conformational changes within the DNA leading to this unusual complex.…”

“…In addition to the 80 amino acid segment, there is conservation in loops that may be involved in strand separation. Loop-2B and Loop-45 are inserted within the separated DNA strands in the cocrystal structure ( 17 ). All of the highlighted proteins have fully conserved residues in Loop-45 (N120, P123, N124, F125, G127, R129 and N132).…”

“…The catalogue of characterized protein-nucleic acid recognition mechanisms is rich and diverse, informed by extensive cocrystal structures. Our prior biochemical work ( 13–14 , 17 ) and the recent CcrM-DNA cocrystal structure shows that CcrM relies on a new recognition mechanism in which the protein induces the unpairing of four out of five base pairs making up the recognition sequence ( 17 ). The potential contribution of this mechanism to the extreme sequence discrimination shown by CcrM further emphasizes its importance ( 14 ).…”

“…The recently described CcrM-DNA cocrystal structure provides insights into a new recognition mechanism, relying on the strand separation of four of the five base pairs within the enzyme’s recognition site ( 17 ). Importantly, this strand displacement goes well beyond the base flipping mechanism observed with other DNA MTases, and unlike CRISPR-Cas9, recognition of the target strand relies solely on interactions with amino acids rather than nucleic acid hybridization.…”