dissection of structure and function of the n-terminal domain of mouse dnmt1 using regional frame-shift mutagenesis解剖结构和功能的鼠标dnmt1的n端结构域使用区域框移诱变.pdfVIP

Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis 1 1 1 1 3 Leonardo D’Aiuto , Marco Marzulli , K. Naga Mohan , Ewa Borowczyk , Federica Saporiti , Andrew 2 1 VanDemark , J. Richard Chaillet * 1 Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 3 Department of Neurology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America Abstract 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