Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI

Youngblood, Benjamin A.; Shieh, Fa Kuen; Rios, Stephanie De Los; Perona, John J.; Reich, Norbert O.

doi:10.1016/j.jmb.2006.07.031

Cited by 13 publications

(24 citation statements)

References 55 publications

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“…A number of M.HhaI studies suggested that the early reaction intermediates form a fast equilibrium [66][67][68][69]. Recent studies showed that a dynamic equilibrium between base flipping and base restacking events can control specificity of other base flipping enzymes [70,71].…”

Section: Target Base Attack Base Flipping and Novel Mechanism-based mentioning

confidence: 99%

Mammalian Cytosine DNA Methyltransferase Dnmt1: Enzymatic Mechanism, Novel Mechanism-Based Inhibitors, and RNA-directed DNA Methylation

Svedružić¹

2008

CMC

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This is a review of the enzymatic mechanism of DNA methyltransferase Dnmt1 and analysis of its implications on regulation of DNA methylation in mammalian cells and design of novel mechanism-based inhibitors. The methylation reaction by Dnmt1 has different phases that depend on DNA substrate and allosteric regulation. Consequently, depending on the phase, the differences in catalytic rates between unmethylated and pre-methylated DNA can vary between 30-40 fold, 3-6 fold or only 1 fold. The allosteric site and the active site can bind different molecules. Allosteric activity depends on DNA sequence, methylation pattern and DNA structure (single stranded vs. double stranded). Dnmt1 binds poly(ADP-ribose) and some RNA molecules. The results on kinetic preferences, allosteric activity and binding preference of Dnmt1 are combined together in one comprehensive model mechanism that can address regulation of DNA methylation in cells; namely, inhibition of DNA methylation by poly(ADP-ribose), RNA-directed DNA methylation by methylated and unmethylated non-coding RNA molecules, and transient interactions between Dnmt1 and genomic DNA. Analysis of reaction intermediates showed that equilibrium between base-flipping and base-restacking events can be the key mechanism in control of enzymatic activity. The two events have equal but opposite effect on accumulation of early reaction intermediates and methylation rates. The accumulation of early reaction intermediates can be exploited to improve the current inhibitors of Dnmt1 and achieve inhibition without toxic modifications in genomic DNA. [1,2-dihydropyrimidin-2-one]-5-methylene-(methylsulfonium)-adenosyl is described as the lead compound. Cell differentiation [1], oncogenic transformation [2], viral infection [3], and long term memory [4] are some of many physiological processes that have created a large interest in DNA methylation. DNA methylation is one of the first steps in epigenetic regulation [5]. Epigenetic regulation is a sequence of molecular events that controls chromatin structure and supports functional organization of eukaryotic genome through cell divisions [6]. DNA methylation is attractive as a fundamental mechanism in functional organization of the human genome, and also as a promising target in development of new drugs for cancer chemotherapy [7,8], suppression of viral infections [3] and possibly mental illness [9]. DNA methylation in mammalian cells is usually associated with five methyltransferases: Dnmt1, Dnmt2, Dnmt3a, Dnmt3b and Dnmt3L. This manuscript will cover Dnmt1, the most predominant DNA methyltransferase in the mammalian cells, and the major target of the current pharmaceutical interest.Dnmt1 is crucial for keeping genomic integrity in higher eukaryotes [6] by preserving existing methylation patterns or by participating in the creation of new methylation patterns. The molecular mechanism that guides these two types of DNA methylation events represent the two most basic questions in DNA methylation research: how new methylation sites are created...

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Section: Target Base Attack Base Flipping and Novel Mechanism-based mentioning

confidence: 99%

Mammalian Cytosine DNA Methyltransferase Dnmt1: Enzymatic Mechanism, Novel Mechanism-Based Inhibitors, and RNA-directed DNA Methylation

Svedružić¹

2008

CMC

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“…SigmaPlot was used to obtain K D DNA values by fitting the data to a rectangular hyperbola curve. 9,11,38 Steady-state and single-turnover assays…”

Section: Dna-binding Affinitymentioning

confidence: 99%

AdoMet-dependent Methyl-transfer: Glu119 Is Essential for DNA C5-Cytosine Methyltransferase M.HhaI

Shieh¹,

Reich²

2007

Journal of Molecular Biology

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“…1A). Populating the closed conformer of the loop is essential for tight DNA binding and stabilizing the target cytosine that is flipped out of the DNA duplex (11)(12)(13). Using stopped-flow fluorescence spectroscopy to monitor the environment of tryptophan (Trp) residues inserted into the catalytic loop, we recently observed reorganization of this loop upon DNA binding in the absence of cofactor using the M.HhaI mutants W41F, W41F/K91W, and W41F/E94W (12).…”

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confidence: 99%

Coupling Sequence-specific Recognition to DNA Modification

Estabrook

Nguyen

Fera

et al. 2009

Journal of Biological Chemistry

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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...

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Engineered Extrahelical Base Destabilization Enhances Sequence Discrimination of DNA Methyltransferase M.HhaI

Cited by 13 publications

References 55 publications

Mammalian Cytosine DNA Methyltransferase Dnmt1: Enzymatic Mechanism, Novel Mechanism-Based Inhibitors, and RNA-directed DNA Methylation

Mammalian Cytosine DNA Methyltransferase Dnmt1: Enzymatic Mechanism, Novel Mechanism-Based Inhibitors, and RNA-directed DNA Methylation

AdoMet-dependent Methyl-transfer: Glu119 Is Essential for DNA C5-Cytosine Methyltransferase M.HhaI

Coupling Sequence-specific Recognition to DNA Modification

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