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

D'Aiuto, L and Marzulli, M and Naga Mohan, K and Borowczyk, E and Saporiti, F and VanDemark, A and Chaillet, JR (2010) Dissection of structure and function of the N-terminal domain of mouse DNMT1 using regional frame-shift mutagenesis. PLoS ONE, 5 (3).

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Deletion analysis of mouse DNMT1, the primary maintenance methyltransferase in mammals, showed that most of the Nterminal 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. © 2010 D'Aiuto et al.


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Item Type: Article
Status: Published
CreatorsEmailPitt UsernameORCID
D'Aiuto, L
Marzulli, Mmam341@pitt.eduMAM341
Naga Mohan, K
Borowczyk, E
Saporiti, F
VanDemark, Aandyv@pitt.eduANDYV0000-0003-3424-4831
Chaillet, JRchaillet@pitt.eduCHAILLET
ContributionContributors NameEmailPitt UsernameORCID
Date: 1 December 2010
Date Type: Publication
Journal or Publication Title: PLoS ONE
Volume: 5
Number: 3
DOI or Unique Handle: 10.1371/journal.pone.0009831
Schools and Programs: Dietrich School of Arts and Sciences > Biological Sciences
Refereed: Yes
MeSH Headings: Animals; DNA (Cytosine-5-)-Methyltransferase--metabolism; DNA, Complementary--metabolism; Embryonic Stem Cells--cytology; Frameshift Mutation; Gene Deletion; Immunohistochemistry--methods; Mice; Mutagenesis; Mutagenesis, Site-Directed; Mutation; Plasmids--metabolism; Protein Folding; Protein Structure, Tertiary; Transcription, Genetic
Other ID: NLM PMC2843745
PubMed Central ID: PMC2843745
PubMed ID: 20352123
Date Deposited: 03 Aug 2012 18:56
Last Modified: 02 Feb 2019 14:55


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