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Through site-specific mutagenesis, three of the ten amino acids of the cytoplasmic domain of the influenza virus hemagglutinin (HA) were individually changed to tyrosines. None of these changes had significant effect on the rate of export, the rate of folding, or the antigenicity of the mutant HAs. However, one of these mutations, substituting tyrosine for(More)
The EcoRV DNA methyltransferase (M.EcoRV) specifically methylates the first adenine within its recognition sequence GATATC. Methylation rates of DNA by this enzyme are strongly influenced by the length of oligonucleotide substrates employed. If substrates >20 bp compared to a 12mer substrate, the kcat/Km increases 100-fold, although the enzyme does not(More)
An assay is described to measure methylation of biotinylated oligonucleotide substrates by DNA methyltransferases using [methyl-3H]-AdoMet. After the methylation reaction the oligonucleotides are immobilized on an avidin-coated microplate. The incorporation of [3H] into the DNA is quenched by addition of unlabeled AdoMet to the binding buffer. Unreacted(More)
Three gene-sized molecules cloned intact from macronuclear DNA served as hybridization probes to study excision of these molecules from chromosomes and their processing during macronuclear development in the hypotrich Euplotes crassus. These molecules occur in integrated forms within polytene chromosomal DNA during macronuclear developmental. After(More)
All DNA methyltransferases (MTases) have similar catalytic domains containing nine blocks of conserved amino acid residues. We have investigated by site-directed mutagenesis the function of 17 conserved residues in the EcoRV alpha-adenine-N6-DNA methyltransferase. The structure of this class of MTases has been predicted recently. The variants were(More)
The EcoRV DNA-(adenine-N6)-methyltransferase (MTase) recognizes GATATC sequences and modifies the first adenine residue within this site. Parts of its DNA interface show high sequence homology to DNA MTases of the dam family which recognize and modify GATC sequences. A phylogenetic analysis of M.EcoRV and dam-MTases suggests that EcoRV arose in evolution(More)
DNA methyltransferases flip their target base out of the DNA helix. Here, we have investigated base flipping by wild-type EcoRV DNA methyltransferase (M.EcoRV) and five M.EcoRV variants (D193A, Y196A, S229A, W231R and Y258A). These variants bind to DNA and S-adenosylmethionine but have a severely reduced catalytic efficiency or are catalytically inactive.(More)