The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. DNA-binding proteins that regulate gene expression play an important biological role and are potentially attractive therapeutic targets. However, the study of their role in different physiological and pathological processes has been hindered by the lack of specific inhibitors. These proteins are especially appealing as drug targets because their ligand is a DNA sequence that can be identified by standard molecular biology techniques and can be synthesized and modified by well established chemistries (1). In addition, recent observations suggest that double-stranded DNA-based oligonucleotides bearing transcription factor recognition sites can be delivered into cells in culture and in vivo and exhibit pharmacological effects (2, 3). A major limitation of this approach is that oligonucleotide antagonists that are identical to the transcription factors cognate site act as stochiometric competitors. Therefore, very high intracellular concentrations are required to effectively engage all the transcription factor available in the cell at all times. An ideal DNA-binding protein antagonist should exhibit higher affinity to the protein than the cognate sequence and bear a slow off rate. In this report we have tested the hypothesis that DNA-based inhibitors of DNA-binding proteins that address these requirements could be developed, using the DNA methyltransferase enzyme (DNA MeTase) 1 as a model DNA-binding protein.Basic oncogenic pathways such as the Ras-Jun signaling pathway have been shown to up-regulate DNA MeTase mRNA (4 -6) and the hyperactivation of DNA MeTase observed in many cancer cells (7,8) occurs in parallel with the development of the aberrant patterns of DNA methylation that these cells exhibit (9, 10). A number of studies suggest that the hyperactivation of DNA MeTase plays a causal role in oncogenesis. For example, the intraperitoneal injection of antisense oligonucleotide to DNA MeTase mRNA into LAF/1 mice bearing tumors derived from the syngeneic tumor cell line Y1 (11) inhibits tumor growth; and in vivo reduction of DNA MeTase levels by either 5-azaCdR treatment or by bearing one mutated allele of DNA MeTase reduces the frequency of appearance of intestinal adenomas in the Min mouse bearing a mutation in the adenomatosis polyposis coli gene (12).Uncovering the biological role of DNA MeTase in vivo requires the a...
DNA-cytosine-5-methyltransferase 1 (DNMT1) is the enzyme believed to be responsible for maintaining the epigenetic information encoded by DNA methylation patterns. The target recognition domain of DNMT1, the domain responsible for recognizing hemimethylated CGs, is unknown. However, based on homology with bacterial cytosine DNA methyltransferases it has been postulated that the entire catalytic domain, including the target recognition domain, is localized to 500 amino acids at the C terminus of the protein. Vertebrate genomes are modified by methylation of ϳ60 -80% of the cytosines residing at the CG dinucleotide sequence (1). The distribution of methylated cytosines is not random, resulting in gene-and tissue-specific patterns of methylation (2). A large body of evidence supports the hypothesis that both methylation patterns and activity of DNA methyltransferases (DNMTs) 1 play critical roles in development and in controlling genome functions such as differential gene expression, chromosome imprinting, and X-chromosome inactivation (3-5). It has also been suggested that DNMT1 is a downstream effector of many oncogenic pathways and a potential target for anticancer therapy (6 -11). We have previously demonstrated that inhibition of DNMT1 leads to an inhibition of DNA replication (12). Recently, it has been shown that DNMT1 is able to form a complex with Rb, E2F, and HDAC1 and repress E2F-responsive expression (13,14). Furthermore, it has been shown that DNMT1 can establish a transcriptional repressive complex with HDAC2 and DMAP1 at replication foci (15). These data suggest that DNMT1 has multiple functions in the cell. However, because the DNMT1 target recognition domain is unknown it is not possible to determine how these multiple functions and protein-protein interactions relate to its target specificity.If DNA methylation patterns contain significant information, there must be a mechanism that ensures its proper inheritance in cell lineages. Razin and Riggs (16) have proposed that patterns of methylation are inherited, because DNMT1 is more proficient in methylating hemimethylated DNA than nonmethylated DNA. This hypothesis has been verified by a number of experiments (17, 18). Another level of specificity is the ability of DNMT1 to recognize CG sequences almost exclusively (19,20). Thus, DNMT1 exhibits both substrate and sequence specificity.The mammalian DNMT1 is a protein postulated to be composed, based on its similarity to other cytosine DNA methyltransferases, of at least three structural components (21-26). These domains are as follows: a catalytic domain at the C terminus, an N-terminal domain that is responsible for localization of the protein to the nucleus and replication foci, and another poorly characterized central domain. It is unclear as yet which segment is responsible for determining its specificity for hemimethylated CG sequences. Previous reports have shown that when the N-terminal domain is cleaved by proteolysis, the enzyme loses its ability to discriminate between hemimethylated and un...
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