Characterisation of independent DNA and multiple Zn-binding domains at the N terminus of human DNA-(cytosine-5) methyltransferase: modulating the property of a DNA-binding domain by contiguous Zn-binding motifs.
We report here a detailed mapping and characterisation of a DNA-binding domain at the N terminus of human DNA-(cytosine-5) methyltransferase. A small region, B1 (codon 202 to 369), was first identified by its Zn- and gross DNA-binding properties. Further fine-mapping using deletion and point mutation analysis shows that the DNA- and Zn-binding domains involve separate peptide motifs, KRRKTTPKEPTEKK (codons 202 to 215) for a bipartite DNA-binding oligopeptide (DB1) and CX2CX13HX2D(X)23EX2EX13CX3H (codons 232 to 297) for possibly two contiguous Zn-binding domains (AZn), which can function independently. However, B1 (containing DB1 and AZn) differs from DB1 because it does not bind to a 30 base-pair duplex. Interestingly, H3 codons 202 to 974, which encloses B1 and B2 (containing the Zn-binding CX2CX2CX4CX2CX2C motif from codon 533 to 550) binds preferentially to 0.8 kb duplexes, as compared with 0.4 to 0.6 kb duplexes. As the homologous murine B1, which targets the murine methylase to replication foci, also binds to DNA and Zn, it is possible that the N terminus of mammalian methylase may be involved in sensing the appropriate length of newly synthesized DNA before methylation by its C terminus. This may enable a time delay for the transient existence of hemi-methylation sites for their unknown biological functions in mammals.
Chuang
L S
LS
Chemical Carcinogenesis Laboratory, Institute of Molecular and Cell Biology National University of Singapore, Republic of Singapore.
Ng
H H
HH
Chia
J N
JN
Li
B F
BF
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D017433:Protein Structure, Secondary; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D015032:Zinc