Coupling Sequence-specific Recognition to DNA Modification

Abstract

Enzymes that modify DNA are faced with significant challenges in specificity for both substrate binding and catalysis. We describe how single hydrogen bonds between M.HhaI, a DNA cytosine methyltransferase, and its DNA substrate regulate the positioning of a peptide loop which is ϳ28 Å away. Stopped-flow fluorescence measurements of a tryptophan inserted into the loop provide real-time observations of conformational rearrangements. These long-range interactions that correlate with substrate binding and critically, enzyme turnover, will have broad application to enzyme specificity and drug design for this medically relevant class of enzymes.Sequence-specific modification of DNA is essential for nearly all forms of life and contributes to a myriad of biological processes including gene regulation, mismatch repair, host defense, DNA replication, and genetic imprinting. Methylation of cytosine and adenine bases is a key epigenetic process whereby phenotypic changes are inherited without altering the DNA sequence (1). The central role of the bacterial and mammalian S-adenosylmethionine (AdoMet) 2 -dependent DNA methyltransferases in virulence regulation and tumorigenesis, respectively, have led these enzymes to be validated targets for antibiotic and cancer therapies (2, 3). However, AdoMet-dependent enzymes catalyze diverse reactions, and the design of potent and selective DNA methyltransferase inhibitors is particularly challenging (4, 5). The design of drugs that bind outside the active site is a particularly attractive means of inhibition for enzymes with common cofactors like AdoMet because off-target inhibition often leads to toxicity (6). Unfortunately, robust methods to identify and characterize such critical binding sites distal from the active site have not been developed.DNA methyltransferases bind to a particular DNA sequence, stabilize the target base into an extrahelical position within the enzyme active site, and transfer the methyl moiety from AdoMet to the DNA (7). During this process, dramatic changes in the DNA structure such as bending, base flipping, or the intercalation of residues into the recognition sequence are often accompanied by large scale protein rearrangements (8).Here we characterized a specific conformational rearrangement of M.HhaI, a model DNA cytosine C 5 methyltransferase with a cognate recognition sequence of 5Ј-GCGC-3Ј. Many structures of M.HhaI are available at high resolution including an ensemble of complexes with either cognate or nonspecific DNA (9, 10). Reorganization of an essential catalytic loop (residues 80 -100) is regulated by sequence-specific protein-DNA interactions that occur ϳ28 Å away from the catalytic loop (Fig. 1). Our work quantifies the importance of such distal communication in sequence-specific DNA modification and provides plausible structural mechanisms.DNA-dependent positioning of the catalytic loop in M.HhaI was first observed crystallographically; cognate DNA stabilizes the loop-closed conformer, while nonspecific DNA leaves the loop in the open c...

Structural Origins of DNA Target Selection and Nucleobase Extrusion by a DNA Cytosine Methyltransferase

“…Together, these structural alterations make the conformation of InC considerably more open at the interface between DNA and the catalytic domain than in ExC, and it is noteworthy that this opening results in partial disassembly of the active site in InC. These structural findings are thus consistent with the results of bio-chemical and biophysical studies on the closely related DCMTase M.HhaI, which showed that DNA binding precedes extrusion of the target nucleobase and initiation of catalysis (8,10,(27)(28)(29). Upon formation of the closed, catalytically active state, the mobile loops of the catalytic domain acquire multiple contacts to both the duplex DNA backbone (Ser-79, Arg-87, Arg-81, Lys-112, and Gln-117) and the extrahelical target cytosine (Gly-68 and Ser-75); these contacts are likely to stabilize the extrahelical, frameshifted conformation of the complex (compare Fig.…”

Direct observation of cytosine flipping and covalent catalysis in a DNA methyltransferase

“…Null or small fluorescence changes were detected when either the DNA or cofator was omitted from the reaction, indicating that the signal is dependent on the formation of the closed ternary complex (4,5,14). The fluorescence signal was also absent in the catalytically active W41F mutant, which contains no tryptophan residues (Supplementary Figure S1).…”

The highly specific, cell cycle–regulated methyltransferase from Caulobacter crescentus relies on a novel DNA recognition mechanism

“…In all cases we used hemimethylated DNA to force one productive binding orientation; in the case of CcrM C. crescentus, the hemimethylated DNA is what the enzyme is normally presented with within the cell. We tested this first with M.HhaI, a structurally characterized DNA cytosine methyltransferase (methylates the underlined cytosines in 5Ј-GCGC-3Ј/5Ј-GCGC-3Ј) (15). As is typical for protein-DNA complexes, the M.HhaI-DNA cocrystal structure shows extensive interactions to bases within both strands, as well to the sugarphosphate backbone of both strands (16).…”