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 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.
Highlights
The mammalian DNA cytosine methyltransferase 1 (DNMT1) is the enzyme primarily responsible for the accurate perpetuation of DNA methylation patterns following cell division
To determine which parts of DNMT1 are required for cellular methyltransferase activity, we generated a collection of cDNAs expressing DNMT1 mutants that differ from each other in the sequence of a stretch of amino acids (Table 1)
As the resultant in-frame insertion-deletion mutations change only a portion of the protein sequence corresponding to the frame-shift, we named this strategy as Regional Frame-Shift Mutagenesis (RFM), and the mutants as RFM mutants
Summary
The mammalian DNA cytosine methyltransferase 1 (DNMT1) is the enzyme primarily responsible for the accurate perpetuation of DNA methylation patterns following cell division. The C-terminal domain (amino acid residues 1148-1620) is characterized by the presence of 10 conserved amino acid motifs, shared with many prokaryotic 5-methyl-cytosine methyltransferases [1]. The more N-terminal subdomain contains the binding site for the DNA methyltransferase associated protein DMAP1 (amino acids 12-105) [2], a functional nuclear localization signal (NLS) (amino acids 191-211), and the binding site for proliferating cell nuclear antigen PCNA (amino acids 162-171) [3]. Loading of DNMT1 onto hemi-methylated DNA is mediated by SRA-domain protein UHRF [4]. The SET and RING associated (SRA) domain of UHRF recognize hemimethylated sites and directs DNMT1 to these sites [4,5]. The C-terminal domain of DNMT1 contains the replication focus targeting sequence (RFTS; amino acids 350-609) [6], the zinc-binding domain, (amino acids 647-693) [7], Bromo Adjacent Homology domain 2 (BAH2; amino acids 968-1104) [8], and two additional NLS (amino acids 259-378 and 630-757) [9]
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