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Published online
doi:10.1083/jcb.200610062
The Journal of Cell Biology, Vol. 176, No. 5, 565-571
The Rockefeller University Press, 0021-9525 $30.00
© Spada et al.
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DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells



Fabio Spada1, Andrea Haemmer1, David Kuch2, Ulrich Rothbauer1, Lothar Schermelleh1, Elisabeth Kremmer3, Thomas Carell2, Gernot Längst4, and Heinrich Leonhardt1

1 Department of Biology II and 2 Department of Chemistry, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany
3 GSF National Research Center for Environment and Health, Institute of Molecular Immunology, 81377 Munich, Germany
4 Institute for Biochemistry, Genetics, and Microbiology, University of Regensburg, 93053 Regensburg, Germany

Correspondence to Heinrich Leonhardt: h.leonhardt{at}lmu.de

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required.

Abbreviations used in this paper: 5-aza-dC, 5-aza-deoxycytidine; DKO, double KO; DNMT, DNA methyltransferase; KO, knockout; PBD, PCNA-binding domain; PCNA, proliferating cell nuclear antigen; wt, wild type.


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