Multiple domains are involved in the targeting of the mouse DNA methyltransferase to the DNA replication foci
Multiple domains are involved in the targeting of the mouse DNA methyltransferase to the DNA replication foci
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DOI：
10.1093/nar/26.4.1038
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发表时间：
1998-02-15
影响因子：
14.9
通讯作者：
Jost, JP
中科院分区：
文献类型：
--
作者：
Liu, YL;Oakeley, EJ;Jost, JP
已经表明，在细胞周期的S期，小鼠DNA甲基转移酶（DNA MTase）通过位于酶的N-末端结构域中的氨基酸序列（aa 207-455）靶向DNA复制位点[Leonhardt，H.，佩奇，A. W.，Weier，H.联合和Bennett，T. H.（1992）Cell，71，855-873]。在本文中，它表明，通过使用增强的绿色荧光蛋白（EGFP）融合，DNA MTase的其他肽序列也参与这种靶向。这项工作的重点是一个序列，下游的报告靶向序列（TS），这是同源的聚溴-1蛋白。该基序（命名为PBHD）通过锌结合基序与报道的靶向序列分开[Bachelor，T. H.（1992）EMBO J，11，2611-2617]。使用着丝粒特异性引物进行的引物原位延伸显示，宿主DNA MTase和含有靶向序列的EGFP融合蛋白都定位于S期晚期和有丝分裂期间的着丝粒区域，但不定位于端粒区域。还发现，在类似于10%的S期细胞中，EGFP融合物不与着丝粒区域共定位。含有这些靶向序列之一或两者的突变体可以作为针对宿主DNA MTase的显性负突变体。EGFP融合蛋白，含有报道的TS（aa 207-455），在整个有丝分裂阶段被靶向到着丝粒区域，这导致发现了内源性DNA MTase的类似行为，尽管宿主MTase显示出比S期细胞中弱得多的染色。DNA MTase在有丝分裂过程中的着丝粒定位的生物学作用目前尚不清楚。
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, 855-873]. 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 similar to 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.