Biochemistry and biological function of mammalian Dnmt1

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DNA methylation is an essential process in mammals involved in several instances of gene regulation. So far, three active DNA methyltransferases have been found in mammals: Dnmt1 involved in maintenance methylation; Dnmt3a and 3b involved in de novo DNA methylation. Dnmt1 is a large protein comprising 1620 amino acid residues. Complete knock-out of the Dnmt1 enzyme is lethal during embryogenesis. Reduction of the amount of Dnmt1 causes demethylation of the DNA in vivo. Furthermore, it leads to an arrest of DNA replication and induces apoptosis. If these effects are due to demethylation of the DNA is not known. Dnmt1 is phosphorylated and interacts with several other proteins in vitro. Biochemical data suggests that it might also be implicated in a process called spreading of methylation, which is often observed in the course of tumor progression. In this application we intend to prepare Dnmt1 variants carrying single or few amino acid exchanges that specifically address defined biochemical functions of the protein and characterize the mutant enzymes in vitro. Subsequently transgenic mice will be generated which contain the same defined exchanges in their endogenous dnmt1 gene to investigate the in vivo function of certain properties of Dnmt1. Following this combined approach, we attempt to investigate a) if the loss of catalytic activity of Dnmt1 is responsible for embryonic lethality of the full knock-out, b) if the interaction of Dnmt1 with the PCNA protein has a role in vivo, c) if phosphorylation of Dnmt1 that has been observed in vitro is of relevance in vivo and d) if an allosteric activation of Dnmt1 for methylation of unmodified DNA that has been observed in vitro and might cause spreading of methylation has a function in vivo. Such experiments which combine site-directed mutagenesis and gene targeting, so far, have not been carried out with any DNA methyltransferase. Finally, we attempt to investigate the biological role of an additional DNA methyltransferase, Dnmt2, whose function so far is not known by preparing a mouse knock-out strain and carefully investigate the transgenic animals over several generations. We expect that the results of these experiments will considerably further our knowledge on DNA methylation and epigenetic gene control.