Peptide Mapping of the Murine DNA Methyltransferase Reveals a Major Phosphorylation Site and the Start of Translation

Abstract

The murine DNA methyltransferase catalyzes the transfer of methyl groups from S-adenosylmethionine to cytosines within d(CpG) dinucleotides. The enzyme is necessary for normal embryonic development and is implicated in a number of important processes, including the control of gene expression and cancer. Metabolic labeling and high pressure liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) were performed on DNA methyltransferase purified from murine erythroleukemia cells. Serine 514 was identified as a major phosphorylation site that lies in a domain required for targeting of the enzyme to the replication foci. These results present a potential mechanism for the regulation of DNA methylation. An essential mechanism for tissue-specific differentiation during embryonic development in mammals is the post-synthetic methylation of d (CpG) 1 dinucleotides in DNA (1). During embryogenesis, 70 -80% of d(CpG) dinucleotides become methylated in a developmentally regulated, tissue-specific fashion (2). The methylation of regulatory DNA elements frequently results in the transcriptional silencing of proximal genes (3). Although no general mechanism for this silencing has been identified, the methylation status of several DNA sequences is known to modulate binding by regulatory proteins (4, 5). HPLC-ESI-MS peptide mapping data demonstratedThe tissue-specific methylation patterns in mammalian cells presumably result from the action of the DNA methyltransferase in concert with other unidentified cellular factors (6), such as chromatin packaging proteins (7), active demethylation systems (8), or particular DNA structures (9) that dictate this enzyme's specificity for certain d(CpG)'s within the genome.The DNA methyltransferase enzyme interacts with p23, a known component of the progesterone receptor complex (10), and it is likely that control of genomic DNA methylation is coupled to receptor-mediated signaling systems in some, as yet unknown, way. A detailed knowledge of the factors involved in the regulation of the methyltransferase enzyme will be necessary to know how genomic methylation patterns are established.The murine DNA methyltransferase is an approximately 180 -190-kDa enzyme that transfers a methyl group from Sadenosylmethionine (AdoMet) onto the C-5 position of cytosine within the d(CpG) dinucleotides of double-stranded DNA. Targeted disruption of the gene for this enzyme is lethal to embryos at the middle stages of gestation (1). A number of functional domains of the enzyme have been identified and are presented in Fig. 1. The catalytic domain lies in the C-terminal third of the protein and contains regions of homology with the prokaryotic DNA methyltransferases, including a conserved AdoMet binding site and catalytic center. The N-terminal twothirds of the enzyme is separated from the catalytic domain by a flexible hinge region consisting of glycine-lysine repeats (11) and contains sequences that bind zinc and DNA independently (11,12). A minor groove DNA-binding motif (SPKK) shown to ...

DNMT1 is a Component of a Multiprotein DNA Replication Complex

“…A serine phosphorylation site has been identified within the replication targeting domain of DNMT1 and could function in this capacity. 55 Both maintenance and de novo methyltransferase activity co-purified with the synthesome complex, and the ration of the two activities (20-30-fold) was similar to that observed for methyltransferase activity in whole cell lysates and for purified recombinant DNMT1. 13,15,28 These data indicate that purification of the replication complex does not lead to the selective concentration of a pool of maintenance methyltransferase activity.…”

A Short DNA Methyltransferase Isoform Restores Methylation In Vivo

“…This conclusion was directly confirmed by protein sequencing of MTase purified from ES cells (see Table I). The same form (MTase ATG3 ) has been identified by peptide mapping in transformed MEL cells (27). We failed to detect any MTase in cell extracts from ES cells, kidney, brain, and thymus with ATG2-Ab, which recognize a peptide sequence between ATG2 and ATG3.…”

“…These data clearly demonstrate that in ES cells, translation of the Dnmt1 mRNA starts at ATG3. Recently, a peptide mapping of MTase from MEL cells was described, which indicated a translation initiation at ATG3 (27). This means that totipotent ES cells and transformed MEL cells contain MTases with identical N termini starting at ATG3 (MTase ATG3 ) with an open reading frame of 1619 amino acids and a calculated molecular mass of 183,034 Da.…”

Preferential Methylation of Unmethylated DNA by Mammalian de Novo DNA Methyltransferase Dnmt3a

“…Although it is known that insect cells do not always reproduce the natural pattern of post-translational modifications seen in mammalian cells (51)(52)(53), a baculovirus system is still regarded as the best alternative (54). It is noteworthy that Dnmt1 was reported to be post-translationally modified in vivo (29,55). The enzymatic activity of recombinant Dnmt3a expressed in Escherichia coli has recently been reported by other groups (26,56).…”