DNA adenine methylation by Caulobacter crescentus Cell Cycle Regulated Methyltransferase (CcrM) is an important epigenetic regulator of gene expression. The recent CcrM-DNA cocrystal structure shows the CcrM dimer disrupts four of the five base pairs of the (5′-GANTC-3′) recognition site. We developed a fluorescence-based assay by which Pyrrolo-dC tracks the strand separation event. Placement of Pyrrolo-dC within the DNA recognition site results in a fluorescence increase when CcrM binds. Non-cognate sequences display little to no fluorescence changes, showing that strand separation is a specificity determinant. Conserved residues in the C-terminal segment interact with the phospho-sugar backbone of the non-target strand. Replacement of these residues with alanine results in decreased methylation activity and changes in strand separation. The DNA recognition mechanism appears to occur with the Type II M.HinfI DNA methyltransferase and an ortholog of CcrM, BabI, but not with DNA methyltransferases that lack the conserved C-terminal segment. The C-terminal segment is found broadly in N4/N6-adenine DNA methyltransferases, some of which are human pathogens, across three Proteobacteria classes, three other phyla and in Thermoplasma acidophilum, an Archaea. This Pyrrolo-dC strand separation assay should be useful for the study of other enzymes which likely rely on a strand separation mechanism.

Strand-separation is emerging as a novel DNA recognition mechanism but the underlying mechanisms and quantitative contribution of strand-separation to fidelity remain obscure. The bacterial DNA adenine methyltransferase, CcrM, recognizes 5′GANTC′3 sequences through a DNA strand-separation mechanism with unusually high selectivity. To explore this novel recognition mechanism, we incorporated Pyrrolo-dC into cognate and noncognate DNA to monitor the kinetics of strand-separation and used tryptophan fluorescence to follow protein conformational changes. Both signals are biphasic and global fitting showed that the faster phase of DNA strand-separation was coincident with the protein conformational transition. Non-cognate sequences did not display strand-separation and methylation was reduced > 300-fold, providing evidence that strand-separation is a major determinant of selectivity. Analysis of an R350A mutant showed that the enzyme conformational step can occur without strand-separation, so the two events are uncoupled. A stabilizing role for the methyl-donor (SAM) is proposed; the cofactor interacts with a critical loop which is inserted between the DNA strands, thereby stabilizing the strand-separated conformation. The results presented here are broadly applicable to the study of other N6-adenine methyltransferases that contain the structural features implicated in strand-separation, which are found widely dispersed across many bacterial phyla, including human and animal pathogens, and some Eukaryotes.

Epigenetic DNA methylation by CcrM C. crescentus is an important regulator of gene expression. CcrM methylates adenine bases on hemimethylated double stranded DNA substrates containing 5′‐GANTC‐3′ sites to regulate gene expression. The recent cocrystal structure shows that CcrM uses a unique DNA recognition mechanism by which four of the five basepairs are completely disrupted. The target strand interacts in base‐specific ways whereas the non‐target strand is largely unperturbed, and does not involve any base‐specific interactions. The dimeric enzyme’s methyltransferase domain and c‐terminal domain interact with the DNA target strand and non‐target strand respectively. Here we present a novel assay that tracks the DNA strand separation step by using a fluorescent probe (Pyrollo‐dC). Placement of Pyrollo‐dC within double stranded DNA at the N position of the CcrM recognition site results in a dramatic increase in fluorescence when CcrM binds; this only occurs when the analog is positioned within the target strand, with much smaller effects involving the non‐target strand. Serine 315 and Histidine 317, which reside in the highly conserved 80‐residue C‐terminal tail, interact with phosphates within the non‐target strand and mutation to alanine results in significant losses of activity and a loss of strand separation, as determine by the Pyrollo‐dC assay. Residue G305A does not interact with the phosphate backbone of the non‐target strand and does show fluorescent signal, suggesting that this mutant is able to strand separate. This mechanism appears to be widespread since other enzymes (e.g., HinfI and an ortholog of CcrM, BabI) exhibit fluorescent signals similar to CcrM. Support or Funding Information NIH Grant