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Research Paper
Phosphorylation of Serine-515 Activates the Mammalian Maintenance Methyltransferase Dnmt1
Rachna Goyal, Philipp Rathert, Heike Laser, Humaira Gowher and Albert Jeltsch
volume 2 | issue 3
july/august/september 2007Subscribe to this journal for $79/year
DNA methyltransferase 1 methylates hemi-methylated CpG sites generated during DNA replication. Serine 515 of this enzyme has been shown to be phosphorylated. To explore the importance of S515 phosphorylation, we generated mutants of Dnmt1 which removed the phosphorylation potential (S515A) or mimic phosphoserine (S515E), purified the proteins from insect cells and analyzed their DNA methylation activity in vitro. The S515E mutant was found to be active, while S515A mutant had severe loss in activity when compared to the wild type protein. The loss of activity of the S515A variant was not due to loss of DNA binding capacity. Furthermore, we show that a phosphorylated peptide whose sequence mimics the surrounding of Ser515 (EKIYISPKIVVE) inhibited the activity of wild type Dnmt1 10-fold more than the non-phosphorylated peptide. The inhibition was specific for Dnmt1 and for the particular peptide sequence. Our data suggest that phosphorylation Ser515 is important for an interaction between the N-terminal domain of Dnmt1 and its catalytic domain that is necessary for activity and that this interaction is specifically disrupted by the phosphorylated peptide. We conclude that phosphorylation of Dnmt1 at Ser515 could be an important regulator of Dnmt1 activity during cell cycle and after proliferative stimuli.
Authors
Rachna Goyal
Institut für Biochemie, Justus-Liebig Universität, Giessen, Germany
Philipp Rathert
School of Engineering and Science, Jacobs University, Bremen, Germany
Heike Laser
School of Engineering and Science, Jacobs University, Bremen, Germany
Humaira Gowher
School of Engineering and Science, Jacobs University Bremen, Bremen, Germany Laboratory of Molecular Biology,National Institutes of Health, Bethesda, Mayland, USA
Albert Jeltsch
School of Engineering and Science, Jacobs University Bremen, Bremen, Germany