Functional analysis of conserved motifs in type III restriction-modification enzymes.
EcoP1I and EcoP15I are members of type III restriction-modification enzymes. EcoPI and EcoP15I DNA methyltransferases transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the N6 position of the second adenine residues in their recognition sequences, 5'-AGACC-3' and 5'-CAGCAG-3' respectively. We have altered various residues in two highly conserved sequences, FxGxG (motif I) and DPPY (motif IV) in these proteins by site-directed mutagenesis. Using a mixture of in vivo and in vitro assays, our results on the mutational analysis of these methyltransferases demonstrate the universal role of motif I in AdoMet binding and a role for motif IV in catalysis. All six cysteine residues in EcoP15I DNA methyltransferase have been substituted with serine and the role of cysteine residues in EcoP15I DNA methyltransferase catalysed reaction assessed. The Res subunits of type III restriction enzymes share a distant sequence similarity with and contain the motifs characteristic of the DEAD box proteins. We have carried out site-directed mutagenesis of the conserved residues in two of the helicase motifs of the EcoP1I restriction enzyme in order to investigate the role of motifs in DNA cleavage by this enzyme. Our findings indicate that certain conserved residues in these motifs are involved in ATP hydrolysis while the other residues are involved in coupling restriction of DNA to ATP hydrolysis. Taken collectively, these results form the basis for a detailed structure-function analysis of EcoP1I and EcoP15I restriction enzymes.
Saha
S
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Department of Biochemistry, Indian Institute of Science, Bangalore.
Ahmad
I
I
Reddy
Y V
YV
Krishnamurthy
V
V
Rao
D N
DN
D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D015263:Deoxyribonucleases, Type III Site-Specific / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D000255:Adenosine Triphosphate; D004247:DNA; D008780:Methyltransferases; C030432:endodeoxyribonuclease EcoP1; C023451:endodeoxyribonuclease EcoP15I; D015263:Deoxyribonucleases, Type III Site-Specific