Peptide Mapping of the Murine DNA Methyltransferase Reveals a Major Phosphorylation Site and the Start of Translation*
- From the Program in Biochemistry and Molecular Biology and Department of Chemistry, University of California, Santa Barbara, California 93106
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.
HPLC-ESI-MS peptide mapping data demonstrated that the purified murine DNA methyltransferase protein contains the N-terminal regions predicted by the recently revised 5′ gene sequences (Yoder, J. A., Yen, R.-W. C., Vertino, P. M., Bestor, T. H., and Baylin, S. B. (1996) J. Biol. Chem. 271, 31092–31097). The evidence suggests a start of translation at the first predicted methionine, with no alternate translational start sites. Our peptide mapping results provide a more detailed structural characterization of the DNA methyltransferase that will facilitate future structure/function studies.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Tel.: 805-893-8368; Fax: 805-893-4120; E-mail: reich{at}sbmm1.ucsb.edu.
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↵1 The abbreviations used are: d(CpG), deoxycytidyl-3′,5′-deoxyguanosine dinucleotide; AdoMet,S-adenosylmethionine; MEL cells, Friend murine erythroleukemia cells; ESI-MS, electrospray ionization mass spectrometry; HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
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↵2 Peptides are denoted using single-letter amino acid nomenclature. The amino acids in bold type are those conserved between various sequences presented (e.g. casein kinase II (27)). The lowercase “p” denotes the confirmed site of phosphorylation.
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↵3 J. Flynn and N. O. Reich, submitted for publication.
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- Received February 11, 1997.
- Revision received April 21, 1997.
