Mutational analysis of conserved residues in HhaI DNA methyltransferase.
Nucleic Acids Res
; 30(12): 2628-38, 2002 Jun 15.
Article
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| MEDLINE
| ID: mdl-12060679
ABSTRACT
HhaI DNA methyltransferase belongs to the C5-cytosine methyltransferase family, which is characterized by the presence of a set of highly conserved amino acids and motifs present in an invariant order. HhaI DNA methyltransferase has been subjected to a lot of biochemical and crystallographic studies. A number of issues, especially the role of the conserved amino acids in the methyltransferase activity, have not been addressed. Using sequence comparison and structural data, a structure-guided mutagenesis approach was undertaken, to assess the role of conserved amino acids in catalysis. Site-directed mutagenesis was performed on amino acids involved in cofactor S-adenosyl-L-methionine (AdoMet) binding (Phe18, Trp41, Asp60 and Leu100). Characterization of these mutants, by in vitro /in vivo restriction assays and DNA/AdoMet binding studies, indicated that most of the residues present in the AdoMet-binding pocket were not absolutely essential. This study implies plasticity in the recognition of cofactor by HhaI DNA methyltransferase.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ADN-Citosina Metilasas
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2002
Tipo del documento:
Article
País de afiliación:
India