5-Methylcytosine-DNA Glycosylase Activity Is Present in a Cloned G/T Mismatch DNA Glycosylase Associated with the Chicken Embryo DNA Demethylation Complex

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 97; no. 10; pp. 5135 - 5139
• Main Authors: Zhu, Bing, Zheng, Yong, Hess, Daniel, Angliker, Herbert, Schwarz, Steffen, Siegmann, Michel, Thiry, Stephane, Jost, Jean-Pierre
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 09-05-2000; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences
• Subjects: 5-Methylcytosine-DNA glycosylase; Amino Acid Sequence; Animals; Base Pair Mismatch; Base Sequence; Biochemistry; Biological Sciences; Chick Embryo; Chickens; Cloning, Molecular; Deoxyribonucleic acid; DNA; DNA glycosylase; DNA Glycosylases; DNA Repair; Embryos; Enzymes; Escherichia coli; Gels; Gene Library; Guanine; Humans; Molecular Sequence Data; N-Glycosyl Hydrolases - chemistry; N-Glycosyl Hydrolases - genetics; N-Glycosyl Hydrolases - metabolism; Oligodeoxyribonucleotides; Oligonucleotides; Poultry; Proteins; Recombinant proteins; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; RNA; RNA helicase; RNA Helicases - metabolism; Sequence Alignment; Sequence Deletion; Sequence Homology, Amino Acid; Sequencing; Thymine; Thymine DNA Glycosylase
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.100107597

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them 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 glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the 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 strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.