MULTIPLE DOMAINS ARE INVOLVED IN THE TARGETING OF THE MOUSE DNA METHYLTRANSFERASE TO THE DNA-REPLICATION FOCI

It has been shown that, during the S-phase of the cell cycle, the mous e DNA methyltransferase (DNA MTase) is targeted to sites of DNA replic ation by an amino acid sequence (aa 207-455) lying in the N-terminal d omain of the enzyme [Leonhardt, H., Page, A. W., Weier, H. U. and Best or, T. H. (1992) Cell, 71, 855-873]. In this paper it is shown, by usi ng enhanced green fluorescent protein (EGFP) fusions, that other pepti de sequences of DNA MTase are also involved in this targeting. The wor k focuses on a sequence, downstream of the reported targeting sequence (TS), which is homologous to the Polybromo-1 protein. This motif (des ignated as PBHD) is separated from the reported targeting sequence by a zinc-binding motif [Bestor, T. H. (1992) EMBO J, 11, 2611-2617]. Pri med in situ extension using centromeric-specific primers was used to s how that both the host DNA MTase and EGFP fusion proteins containing t he targeting sequences were localized to centromeric, but not telomeri c, regions during late S-phase and mitosis. Also found was that, in si milar to 10% of the S-phase cells, the EGFP fusions did not co-localiz e with the centromeric regions. Mutants containing either, or both, of these targeting sequences could act as dominant negative mutants agai nst the host DNA MTase. EGFP fusion proteins, containing the reported TS (aa 207-455), were targeted to centromeric regions throughout the m itotic 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.