Multiple domains are involved in the targeting of the mouse DNA methyltransferase to the DNA replication foci

Abstract

It has been shown that, during the S-phase of the cell cycle, the mouse DNA methyltransferase (DNA MTase) is targeted to sites of DNA replication by an amino acid sequence (aa 207-455) lying in the N-terminal domain of the enzyme [Leonhardt, H., Page, A. W., Weier, H. U. and Bestor, T. H. (1992) Cell , 71, 865-873]. In this paper it is shown, by using enhanced green fluorescent protein (EGFP) fusions, that other peptide sequences of DNA MTase are also involved in this targeting. The work focuses on a sequence, downstream of the reported targeting sequence (TS), which is homologous to the Polybromo-1 protein. This motif (designated as PBHD) is separated from the reported targeting sequence by a zinc-binding motif [Bestor , T. H. (1992) EMBO J , 11, 2611-2617]. Primed in situ extension using centromeric-specific primers was used to show that both the host DNA MTase and EGFP fusion proteins containing the targeting sequences were localized to centromeric, but not telomeric, regions during late S-phase and mitosis. Also found was that, in approximately 10% of the S-phase cells, the EGFP fusions did not co-localize with the centromeric regions. Mutants containing either, or both, of these targeting sequences could act as dominant negative mutants against the host DNA MTase. EGFP fusion proteins, containing the reported TS (aa 207-455), were targeted to centromeric regions throughout the mitotic stage which lead to the discovery of a similar behavior of the endogenous DNA MTase although the host MTase showed much less intense staining than in S-phase cells. The biological role of the centromeric localization of DNA MTase during mitosis is currently unknown.

Global delay in nascent strand DNA methylation

“…For CpG methylation, the rapid initial surge that we observe is consistent with the replication fork recruiting DNMT1 to newly synthesized DNA via its PCNA binding domain, which, when absent, reduces nascent strand methylation rate as previously noted 31 . It appears that the initial seeding of DNMT1 generally does not occur at fixed locations, as we were not able to identify commonly or disproportionately hypermethylated features in our pulse-chase experiment.…”

Expression of murine DNA methyltransferases Dnmt1 and Dnmt3a in the yeast Saccharomyces cerevisiae

“…Eukaryotic DNA methyltransferases interact, through their N-terminal non-catalytic domains, with numerous nuclear components (Bestor, 2000;Chuang et al, 1997;Hermann et al, 2004;Liu et al, 1998;Robertson et al, 2000;Rountree et al, 2000). It was thus likely that introducing such interactors into the yeast cell nucleus could disturb nuclear homeostasis and affect cell functioning.…”

“…Consequently, abundant data have accumulated documenting complex functional interactions of the DNA methyltransferases with each other, with their accessory factors and with other nuclear components (e.g. Chuang et al, 1997;Liu et al, 1998;Robertson et al, 2000;Rountree et al, 2000). This complexity of the molecular machinery determining the pattern of DNA methylation is undoubtedly the chief reason for our relatively poor understanding of the intricacies of the process.…”

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

“…Such a structural organization supposes that the Dnmt1 gene has arisen as fusion of a MTase gene with nonhomologous genes of other DNA-binding proteins [11,83]. It is worth to note that Dnmt1 targeting to replication foci is provided by three types of domains located in its N-terminal part: the PCNA binding domain, the RFTS domain, and the BAH domains [84][85][86]. Studying of Dnmt1 deletion mutants revealed 3 different DNA binding regions: the residues 1-343, the CXXC domain (residues 613-748), and the catalytic domain (residues 1124-1620) [13].…”