UC Santa Barbara

DNA methyltransferases are responsible for transcriptional regulation, cell cycle progression, DNA repair, DNA protection, tumor suppression, and several other important biological processes. Aberrant bacterial DNA methylation can lead to cell death and loss of protection against viral infection; in humans this leads to cancer, autoimmune diseases, metabolic disorders, and neurological disorders. Thus, DNA methyltransferases are common drug targets for cancer therapeutics and novel antibiotics.The conformational transitions in DNA and protein that govern recognition, substrate accessibility, and catalysis are fundamental to understanding the mechanisms that regulate biological processes. The bacterial N6-adenine cell-cycle regulated DNA methyltransferase, CcrM, is the first DNA methyltransferase shown to rely on a unique DNA recognition mechanism in which the DNA strands are separated and most recognition interactions appear to involve the target strand. Strand-separation is emerging as a novel DNA recognition mechanism but the underlying mechanisms and quantitative contribution of strand-separation to fidelity remain obscure for any enzyme (CRISPR-Cas9 and RNA polymerase sigma factor). This work uncovers the fundamental steps governing CcrM's DNA strand separation and high-fidelity DNA recognition mechanism. We relied on mutational analysis of highly conserved residues in the C-terminal domain, Loop-2B, Loop-45, and the active site to probe the function of structurally implicated protein moieties. We collected stopped-flow kinetic fluorescence to monitor transitions in DNA and protein and relied on rigorous global data fitting to understand the states that regulate catalysis in CcrM. 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 (C-term domain) 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. Loops 2B and 45 are inserted between the strand-separated DNA interface. During strand-separation, residues within Loops 2B, 45, and 6E contact the target DNA strand that undergoes methylation. R44 and R129 (Loop-2B and Loop-45, respectively) when mutated to Alanine, disrupt strand-separation and are catalytically inactive. The highly conserved Loop-45 residue F125, which is positioned between the separated DNA strands, is also essential for maintaining the strand-separated intermediate; replacement of F125 with Alanine, Leucine, and Tryptophan results in various perturbations of strand-separation that are correlated to the bulkiness of the substituted residue. Global fitting for each mutant shows that generation and stabilization of DNA strand-separation are perturbed, providing a functional role for these loops in generating and stabilizing the strand-separated intermediate, which is essential for discrimination and catalysis. Employing a fluorescent adenine analog (6MAP) at the target position to monitor base flipping, we resolved that target adenine base flipping follows DNA strand separation and is followed by fast methylation and fast product DNA release. A W57F mutant (active site) displayed an unaltered rate of base flipping as monitored by 6MAP fluorescence but greatly reduced rate of methylation, showing that base-flipping and methylation can be uncoupled. In addition, single-stranded DNA bypasses the DNA strand separation step, while rates of base flipping measured by 6MAP fluorescence and DNA methylation are similar to dsDNA. Global data fitting for each model resolves that base flipping of the target adenine is the rate-limiting step in catalysis. The results presented here are broadly applicable to the study of other N6-adenine methyltransferases that contain the structural moieties implicated in strand-separation (Loop-2B, Loop-45, and the C-terminal domain), which are found widely dispersed across many bacterial phyla, including human and animal pathogens. Insights into CcrM’s mechanism of DNA strand-separation are likely to clarify strand-separation mechanisms for other enzymes such as CRISPR-Cas9 and RNA polymerase sigma factor. Additionally, the elevated understanding of CcrM’s strand-separation mechanism could be useful for the development of selective CcrM inhibitors as novel antibiotics.