5-methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence

Citation
B. Zhu et al., 5-methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence, NUCL ACID R, 28(21), 2000, pp. 4157-4165
Citations number
21
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
NUCLEIC ACIDS RESEARCH
ISSN journal
0305-1048 → ACNP
Volume
28
Issue
21
Year of publication
2000
Pages
4157 - 4165
Database
ISI
SICI code
0305-1048(20001101)28:21<4157:5DGAIA>2.0.ZU;2-0
Abstract
A 1468 bp cDNA coding for the chicken homolog of the human MBD4 GTT mismatc h DNA glycosylase was isolated and sequenced. The derived amino acid sequen ce (416 amino acids) shows 46% identity with the human MBD4 and the conserv ed catalytic region at the C-terminal end (170 amino acids) has 90% identit y. The non-conserved region of the avian protein has no consensus sequence for the methylated DNA binding domain. The recombinant proteins from human and chicken have GTT mismatch as well as 5-methylcytosine (5-MeC) DNA glyco sylase activities. When tested by gel shift assays, human recombinant prote in with or without the methylated DNA binding domain binds equally well to symmetrically, hemimethylated DNA and non-methylated DNA. However, the enzy me has only 5-MeC DNA glycosylase activity with the hemimethylated DNA. Foo tprinting of human MBD4 and of an N-terminal deletion mutant with partially depurinated and depyrimidinated substrate reveal a selective binding of th e proteins to the modified substrate around the CPG. As for 5-MeC DNA glyco sylase purified from chicken embryos, MBD4 does not use oligonucleotides co ntaining mCpA, mCpT or mCpC as substrates. An mCpG within an A+T-rich oligo nucleotide is a much better substrate than an A+T-poor sequence. The K-m of human MBD4 for hemimethylated DNA is similar to 10(-7) M with a V-max of s imilar to 10(-11) mol/h/mug protein. Deletion mutations show that GTT misma tch and 5-MeC DNA glycosylase are located in the C-terminal conserved regio n. In sharp contrast to the 5-MeC DNA glycosylase isolated from the chicken embryo DNA demethylation complex, the two enzymatic activities of MBD4 are strongly inhibited by RNA. In situ hybridization with antisense RNA indica te that MBD4 is only located in dividing cells of differentiating embryonic tissues.