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Cloning and Characterization of a New Site-Specific Methyl-Directed ElmI Endonuclease Recognizing and Cleaving C5-methylated DNA Sequence 5'-G(5mC)^NG(5mC)-3'.

Chernukhin VA, Gonchar DA, Abdurashitov MA, Belichenko OA, Dedkov VS, Mikhnenkova NA, Lomakovskaya EN, Udal'yeva SG, Degtyarev SKh - Acta Naturae (2016 Jan-Mar)

Bottom Line: Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI.It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5'-GCNGC- 3' before the central nucleotide "N" if this sequence contains two 5-methylcytosines.However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated.

View Article: PubMed Central - PubMed

Affiliation: SibEnzyme, Timakova St., 2/12, 630117, Novosibirsk, Russia.

ABSTRACT
Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI. A highly conserved DNA primary structure of these open reading frames in different genera of Enterobacteria (Escherichia, Klebsiella and Cronobacter) has allowed researchers to create primers for PCR screening, which was carried out on Enterobacteria DNA collected from natural sources. The DNA fragment, about 440 bp in length, was amplified by use of the genomic DNA of a wild E.coli LM N17 strain as a template and was inserted into the pMTL22 vector. Endonuclease activity was detected in an E.coli ER 2267 strain transformed with the obtained construction. A new enzyme named ElmI was purified by chromatographic techniques from the recombinant strain biomass. It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5'-GCNGC- 3' before the central nucleotide "N" if this sequence contains two 5-methylcytosines. However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated.

No MeSH data available.


Related in: MedlinePlus

Alignment of the amino acid sequences of BisI and ElmI with the most homologousproteins from enterobacteria. The designations of amino acid sequencescorrespond to GenBank numbers, as described in the text. Amino acids that areidentical in all presented protein sequences are shown in white on a blackbackground. Amino acids with similar physical and chemical properties are shownin black on a gray background.
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Figure 1: Alignment of the amino acid sequences of BisI and ElmI with the most homologousproteins from enterobacteria. The designations of amino acid sequencescorrespond to GenBank numbers, as described in the text. Amino acids that areidentical in all presented protein sequences are shown in white on a blackbackground. Amino acids with similar physical and chemical properties are shownin black on a gray background.

Mentions: Figure 1 showsmultiple alignment of the amino acid sequencesof the four highly homologous enterobacterial proteins from the first group,which have a BisI sequence. The sequence of endonuclease ElmI, which wasdetected by PCR screening, is also shown (see the description below).


Cloning and Characterization of a New Site-Specific Methyl-Directed ElmI Endonuclease Recognizing and Cleaving C5-methylated DNA Sequence 5'-G(5mC)^NG(5mC)-3'.

Chernukhin VA, Gonchar DA, Abdurashitov MA, Belichenko OA, Dedkov VS, Mikhnenkova NA, Lomakovskaya EN, Udal'yeva SG, Degtyarev SKh - Acta Naturae (2016 Jan-Mar)

Alignment of the amino acid sequences of BisI and ElmI with the most homologousproteins from enterobacteria. The designations of amino acid sequencescorrespond to GenBank numbers, as described in the text. Amino acids that areidentical in all presented protein sequences are shown in white on a blackbackground. Amino acids with similar physical and chemical properties are shownin black on a gray background.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837579&req=5

Figure 1: Alignment of the amino acid sequences of BisI and ElmI with the most homologousproteins from enterobacteria. The designations of amino acid sequencescorrespond to GenBank numbers, as described in the text. Amino acids that areidentical in all presented protein sequences are shown in white on a blackbackground. Amino acids with similar physical and chemical properties are shownin black on a gray background.
Mentions: Figure 1 showsmultiple alignment of the amino acid sequencesof the four highly homologous enterobacterial proteins from the first group,which have a BisI sequence. The sequence of endonuclease ElmI, which wasdetected by PCR screening, is also shown (see the description below).

Bottom Line: Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI.It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5'-GCNGC- 3' before the central nucleotide "N" if this sequence contains two 5-methylcytosines.However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated.

View Article: PubMed Central - PubMed

Affiliation: SibEnzyme, Timakova St., 2/12, 630117, Novosibirsk, Russia.

ABSTRACT
Putative open reading frames of MD-endonucleases have been identified in Enterobacteria genomes as a result of the search for amino acid sequences homologous to MD-endonuclease BisI. A highly conserved DNA primary structure of these open reading frames in different genera of Enterobacteria (Escherichia, Klebsiella and Cronobacter) has allowed researchers to create primers for PCR screening, which was carried out on Enterobacteria DNA collected from natural sources. The DNA fragment, about 440 bp in length, was amplified by use of the genomic DNA of a wild E.coli LM N17 strain as a template and was inserted into the pMTL22 vector. Endonuclease activity was detected in an E.coli ER 2267 strain transformed with the obtained construction. A new enzyme named ElmI was purified by chromatographic techniques from the recombinant strain biomass. It was discovered that similarly to BisI this enzyme specifically cleaves the methylated DNA sequence 5'-GCNGC- 3' before the central nucleotide "N" if this sequence contains two 5-methylcytosines. However, unlike BisI, ElmI more efficiently cleaves this sequence if more than two cytosine residues are methylated.

No MeSH data available.


Related in: MedlinePlus