5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex

Citation
B. Zhu et al., 5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex, P NAS US, 97(10), 2000, pp. 5135-5139
Citations number
31
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
ISSN journal
0027-8424 → ACNP
Volume
97
Issue
10
Year of publication
2000
Pages
5135 - 5139
Database
ISI
SICI code
0027-8424(20000509)97:10<5135:5GAIPI>2.0.ZU;2-U
Abstract
We previously have shown that DNA demethylation by chicken embryo 5-methylc ytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of t hem are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylas e was isolated and sequenced. The derived amino acid sequence (408 aa) show s 80% identity with the human G/T mismatch DNA grycosylase. and both the C and N-terminal parts have about 50% identity. As for the highly purified ch icken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombin ant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylat ed CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for th e recombinant protein expressed in E. coli and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme st rongly inhibits G/T mismatch glycosylase while slightly stimulating the act ivity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG a ctivities share the same areas of the N- and C-terminal parts of the protei n. in reconstitution experiments RNA helicase in the presence of recombinan t RNA and ATP potentiates the activity of 5-MCDG.