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Cloning of the Dam methyltransferase gene from Haemophilus influenzae bacteriophage HP1
1Institute of Microbiology, Warsaw University, Poland.
Abstract:
The putative product of orf13 from the genome of Haemophilus influenzae HP1 bacteriophage shows homology only to bacteriophage T1 Dam methyltransferase, and a weak similarity to the conserved amino acids sequence motifs characteristic of m6A-methyltransferases. Especially interesting is lack of characteristic motif I responsible for binding of S-adenosylmethionine. Despite this fact, a DNA sequence of HP1 bacteriophage of Haemophilus influenzae encoding methyltransferase activity was cloned and expressed in Escherichia coli using pMPMT4 omega expression vector. The cloned methyltransferase recognizes the sequence 5'-GATC-3' and methylates an adenine residue. The enzyme methylates both double- and single-stranded DNA substrates.
Insights
Researchers identified a novel DNA methyltransferase from Haemophilus influenzae HP1 bacteriophage. This enzyme, despite lacking a key motif, effectively methylates adenine in GATC sequences on various DNA types.
Area of Science:
- Bacteriophage genetics
- Enzymology
- Molecular biology
Background:
- The Haemophilus influenzae HP1 bacteriophage orf13 gene product exhibits limited homology to known DNA methyltransferases.
- Notably, it lacks the conserved motif typically responsible for S-adenosylmethionine binding, a crucial cofactor for methyltransferases.
Purpose of the Study:
- To clone and express the methyltransferase activity encoded by the HP1 bacteriophage orf13 gene.
- To characterize the substrate specificity and activity of the novel methyltransferase.
Main Methods:
- Cloning and expression of the orf13 gene in Escherichia coli using the pMPMT4 omega expression vector.
- Biochemical assays to determine DNA methyltransferase activity and substrate recognition.
Main Results:
- The cloned enzyme exhibits methyltransferase activity, recognizing the 5'-GATC-3' sequence.
- The enzyme successfully methylates adenine residues in both double-stranded and single-stranded DNA substrates.
- The methyltransferase functions despite the absence of the canonical S-adenosylmethionine-binding motif.
Conclusions:
- The HP1 bacteriophage encodes a functional DNA methyltransferase with unique characteristics.
- This enzyme represents a novel class of methyltransferases, expanding our understanding of DNA modification systems in bacteriophages.