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Novel non-specific DNA adenine methyltransferases.

Drozdz M, Piekarowicz A, Bujnicki JM, Radlinska M - Nucleic Acids Res. (2011)

Bottom Line: Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes.Their potential 'sequence specificity' could be summarized as AB or BA (where B = C, G or T).Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.

ABSTRACT
The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N(6)-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N(6)-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N(6)-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential 'sequence specificity' could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation.

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Comparison of initial velocities of methylation reactions of oligonucleotide duplexes (Supplementary Table S2) carried out by the Hia5 and Hin1523 enzymes.
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gkr1039-F3: Comparison of initial velocities of methylation reactions of oligonucleotide duplexes (Supplementary Table S2) carried out by the Hia5 and Hin1523 enzymes.

Mentions: Substrates with repetitions of the dinucleotides CA and GA (CA10, GA10, respectively) were methylated with comparable rates by Hia5, and TA was methylated slightly slower (Figure 3). The dinucleotide CA was methylated by the Hin1523 enzyme with the highest rate, TA was methylated slightly slower and GA two times slower than two others (Figure 3). The poly-dA/poly-dT duplex was not methylated by either the Hia5 or Hin1523 enzymes. To exclude the nature of the sequence (tract character) and potential duplex instability as the cause of this negative result, we tested substrates containing one or three adenines flanked by m6As i.e. m6A A m6A (Cm2A1) and m6A AAA m6A (Cm2A3). These duplexes were also not methylated by Hia5 and Hin1523.Figure 3.


Novel non-specific DNA adenine methyltransferases.

Drozdz M, Piekarowicz A, Bujnicki JM, Radlinska M - Nucleic Acids Res. (2011)

Comparison of initial velocities of methylation reactions of oligonucleotide duplexes (Supplementary Table S2) carried out by the Hia5 and Hin1523 enzymes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299994&req=5

gkr1039-F3: Comparison of initial velocities of methylation reactions of oligonucleotide duplexes (Supplementary Table S2) carried out by the Hia5 and Hin1523 enzymes.
Mentions: Substrates with repetitions of the dinucleotides CA and GA (CA10, GA10, respectively) were methylated with comparable rates by Hia5, and TA was methylated slightly slower (Figure 3). The dinucleotide CA was methylated by the Hin1523 enzyme with the highest rate, TA was methylated slightly slower and GA two times slower than two others (Figure 3). The poly-dA/poly-dT duplex was not methylated by either the Hia5 or Hin1523 enzymes. To exclude the nature of the sequence (tract character) and potential duplex instability as the cause of this negative result, we tested substrates containing one or three adenines flanked by m6As i.e. m6A A m6A (Cm2A1) and m6A AAA m6A (Cm2A3). These duplexes were also not methylated by Hia5 and Hin1523.Figure 3.

Bottom Line: Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes.Their potential 'sequence specificity' could be summarized as AB or BA (where B = C, G or T).Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Virology, Institute of Microbiology, Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.

ABSTRACT
The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N(6)-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N(6)-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N(6)-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential 'sequence specificity' could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation.

Show MeSH
Related in: MedlinePlus