Dissection of structure and function of the N-terminal domain of mouse DNMT1 using regional frame-shift mutagenesis

• Published in: PloS one 2010-03, Vol.5 (3), p.e9831
• Main Authors: D'Aiuto, Leonardo, Marzulli, Marco, Mohan, K Naga, Borowczyk, Ewa, Saporiti, Federica, Vandemark, Andrew, Chaillet, J Richard
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Amino acids; Animals; Binding sites; Deletion mutant; Developmental Biology/Stem Cells; Directed evolution; Dissection; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases - metabolism; DNA methylation; DNA, Complementary - metabolism; DNMT1 protein; Embryonic Stem Cells - cytology; Enzymatic activity; Evolutionary design method; Frameshift Mutation; Gene Deletion; Genetics and Genomics/Epigenetics; Genetics and Genomics/Gene Expression; Genetics and Genomics/Gene Function; Immunohistochemistry - methods; Methylation; Methyltransferase; Methyltransferases; Mice; Molecular Biology/Chromatin Structure; Molecular Biology/DNA Methylation; Mutagenesis; Mutagenesis, Site-Directed; Mutants; Mutation; Plasmids - metabolism; Protein Folding; Protein structure; Protein Structure, Tertiary; Regional analysis; Regional development; Stem cells; Structural stability; Structure-function relationships; Surgery; Transcription, Genetic
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0009831

Deletion analysis of mouse DNMT1, the primary maintenance methyltransferase in mammals, showed that most of the N-terminal regulatory domain (amino acid residues 412-1112) is required for its enzymatic activity. Although analysis of deletion mutants helps to identify regions of a protein sequence required for a particular activity, amino acid deletions can have drastic effects on protein structure and/or stability. Alternative approaches represented by rational design and directed evolution are resource demanding, and require high-throughput selection or screening systems. We developed Regional Frame-shift Mutagenesis (RFM) as a new approach to identify portions required for the methyltransferase activity of DNMT1 within the N-terminal 89-905 amino acids. In this method, a short stretch of amino acids in the wild-type protein is converted to a different amino acid sequence. The resultant mutant protein retains the same amino acid length as the wild type, thereby reducing physical constrains on normal folding of the mutant protein. Using RFM, we identified three small regions in the amino-terminal one-third of the protein that are essential for DNMT1 function. Two of these regions (amino acids 124-160 and 341-368) border a large disordered region that regulates maintenance methylation activity. This organization of DNMT1's amino terminus suggests that the borders define the position of the disordered region within the DNMT1 protein, which in turn allows for its proper function.