5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex.
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.
Zhu
B
B
Friedrich Miescher-Institut, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.
Zheng
Y
Y
Hess
D
D
Angliker
H
H
Schwarz
S
S
Siegmann
M
M
Thiry
S
S
Jost
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D020137:Base Pair Mismatch; D001483:Base Sequence; D002642:Chick Embryo; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D015723:Gene Library; D006147:Guanine; D006801:Humans; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D020365:RNA Helicases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013941:Thymine; D044502:Thymine DNA Glycosylase
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006147:Guanine; C093335:5-methylcytosine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D044502:Thymine DNA Glycosylase; C113921:mismatch-specific thymine uracil-DNA glycosylase; D020365:RNA Helicases; D013941:Thymine