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J. Biol. Chem., Vol. 276, Issue 45, 41991-41997, November 9, 2001
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From the Purification from calf thymus of a DNA
N-glycosylase activity (HMUDG) that released
5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus
subtilis phage SPO1 was undertaken. Analysis of the most
purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to
identify HMUDG by inspection. Therefore, we renatured the enzyme after
SDS-polyacrylamide gel electrophoresis and assayed slices of the gel
for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or
identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase
(hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a
recombinant glutathione S-transferase fusion protein that
was shown to release 5hmUra with 20× the specific activity of the most
purified bovine fraction. We conclude that hSMUG1 and HMUDG are the
same protein.
Definitive Identification of Mammalian 5-Hydroxymethyluracil DNA
N-Glycosylase Activity as SMUG1*
,,,,
Department of Pathology and Kaplan
Comprehensive Cancer Center, the § Department of
Pharmacology and Skirball Institute of Biomolecular Medicine, and the
¶ Department of Cell Biology, New York University School of
Medicine, New York, New York 10016
*
This work was supported by National Institutes of Health
Grants CA 16669 and CA 49869 (to G. W. T.), CA 16087 (to Kaplan
Comprehensive Cancer Center) and 5T32 CA-09161 (to D. R. M.), and a
Whitehead Presidential Fellowship (to T. A. N.).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.
To whom correspondence should be addressed: Dept. of
Pathology, New York University Medical Center, 550 First Ave., New
York, NY 10016. Tel.: 212-263-5473; Fax: 212-263-8211; E-mail:
george.teebor@med.nyu.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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