03 Fakultät Chemie
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/4
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Item Open Access Regulation of the catalytic activity and specificity of DNA nucleotide methyltransferase 1(2014) Bashtrykov, Pavel; Jeltsch, Albert (Prof. Dr.)DNA nucleotide methyltransferase 1 (Dnmt1) is mainly responsible for the maintenance of DNA methylation in mammals and plays a crucial role in the epigenetic control of gene expression. Dnmt1 recognizes and methylates hemimethylated CpG sites formed during DNA replication. In the present work, the mechanistic details of the substrate recognition by the catalytic domain of Dnmt1, the possible role of the CXXC and RFTS domains of Dnmt1 in the regulation of specificity and activity of Dnmt1, and the influence of the Ubiquitin-like PHD and RING finger domain-containing 1 (Uhrf1) protein on the enzymatic properties of Dnmt1 was investigated. Using modified substrates, the functional roles of individual contacts of the Dnmt1 catalytic domain with the CpG site of the DNA substrate were analysed. The data show that the interaction with the 5-methylcytosine:guanine pair is required for the catalytic activity of Dnmt1, whereas the contacts to the non-target strand guanine are not important, since its replacement with adenine increased the activity of Dnmt1. It was proposed that the CXXC domain binding to unmethylated CpG sites increases the specificity of Dnmt1 for hemimethylated DNA. Our data showed that the CXXC domain does not influence the enzyme’s specificity in the full-length Dnmt1. In contrast, mutagenesis in the catalytic domain introducing an M1235S exchange resulted in a significant reduction in specificity. Therefore, the readout for the hemimethylated DNA occurs within its catalytic domain. It was observed in a crystal structure that the RFTS domain of Dnmt1 inhibits the activity of the enzyme by binding to the catalytic domain and blocking the entry of the DNA. By amino acid substitution in the RFTS domain its positioning within the catalytic domain was destabilized and a corresponding increase in the catalytic rate was observed, which supports this concept and suggests a possible mechanism to allosterically regulate the activity of Dnmt1 in cells. Uhrf1 has been shown to target Dnmt1 to replicated DNA, which is essential for DNA methylation. Here it is demonstrated that Uhrf1 as well as its isolated SRA domain increase the activity and specificity of Dnmt1 in an allosteric mechanism. The stimulatory effect was independent of the SRA domain’s ability to bind hemimethylated DNA. The RFTS domain of Dnmt1 is required for the stimulation, since its deletion or blocking of its interaction with the SRA domain, significantly reduced the ability of Uhrf1 to increase the activity and specificity of Dnmt1. Uhrf1, therefore, plays multiple roles that support DNA methylation including targeting of Dnmt1, its stimulation and an increase of its specificity.