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

Abstract

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...

Decrease in catalytic capacity of γ-secretase can facilitate pathogenesis in sporadic and Familial Alzheimer's disease

“…Similar enzyme-based approach in studies of DNA methyltransferase lead to description of different regulation mechanisms almost 10 years before the initial ideas become the mainstream (Svedruzic, 2008;Svedruzic andReich, 2005a,2005b;Di Ruscio et al, 2013).…”

Diverse Domains of (Cytosine-5)-DNA Methyltransferases: Structural and Functional Characterization

“…The mutations derived from the cytosine deamination might not be lethal for a bacterial cell and could help to prevent hydrolysis of cellular DNA by phage endonucleases. So, the ability of a C5-DNA MTase to catalyze deamination can turn out a physiological advantage for a bacterial cell [5]. On the contrary, mammalian cells are not likely to benefit from this kind of mutagenesis and therefore have developed mechanisms which provide low mutagenesis rate.…”

“…To date, the structural characteristics of the catalytic domain from different MTases, the details of the catalytic mechanism and the biological functions of C5-DNA MTases from different organisms are summarized in a variety of reviews (for example, see [1,[4][5][6][7][8]). Therefore, these aspects are discussed here rather briefly.…”

In silico design of the first DNA-independent mechanism-based inhibitor of mammalian DNA methyltransferase Dnmt1

“…Dnmt1 is the principal DNA methyltransferase in mammalian cells [6, 12]. More than 20 different inhibitors of mammalian Dnmt1 have been described in the last 30 years [13].…”

“…The cytosine derivatives have been very useful in mechanistic studies of DNA methyltransferases [18–20], however their full applicability as inhibitors of DNA methylation is very limited due to high toxicity in cells [13]. Very little improvement has been introduced following the initial studies, mostly due to the unusually challenging mechanism of the target base attack in the process of DNA methylation [12, 18–22]. Consistent with earlier activity studies recent crystal structures showed that the target base attack depends on multiple flexible loops in the protein structure [18–21, 23].…”