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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

Incomplete cleavage of pFsp4HI3/DriI showing higher ElmI activity in5’-GCNGC-3’ sequences with three or four 5-methylcytosines,compared with 5’-GCNGC-3 sequences with only two 5-methylcytosines thatBisI cuts preferably. Electrophoresis in 1% agarose gel. Lanes: 1-8,pFsp4HI3/DriI, treated with serial 2-fold dilutions of ElmI (initial amount,added to the initial reaction mixture is 1 u.a.); 9, DNA molecular weightmarker 1 kb; 10-17, pFsp4HI3/DriI, treated with serial 2-fold dilutions of BisI(initial amount, added to the reaction mixture is 1 u.a.); 18, pFsp4HI3/DriI.
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Figure 4: Incomplete cleavage of pFsp4HI3/DriI showing higher ElmI activity in5’-GCNGC-3’ sequences with three or four 5-methylcytosines,compared with 5’-GCNGC-3 sequences with only two 5-methylcytosines thatBisI cuts preferably. Electrophoresis in 1% agarose gel. Lanes: 1-8,pFsp4HI3/DriI, treated with serial 2-fold dilutions of ElmI (initial amount,added to the initial reaction mixture is 1 u.a.); 9, DNA molecular weightmarker 1 kb; 10-17, pFsp4HI3/DriI, treated with serial 2-fold dilutions of BisI(initial amount, added to the reaction mixture is 1 u.a.); 18, pFsp4HI3/DriI.

Mentions: In order to establish how effectively ElmI cleaves a 5’-GCNGC-3’sequence with a higher number of 5-methylcytosine residues, pFsp4HI3/DriI wastreated with ElmI in an amount insufficient for complete hydrolysis and theresult was compared with the result of its cleavage by BisI endonuclease(Figure 4).


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)

Incomplete cleavage of pFsp4HI3/DriI showing higher ElmI activity in5’-GCNGC-3’ sequences with three or four 5-methylcytosines,compared with 5’-GCNGC-3 sequences with only two 5-methylcytosines thatBisI cuts preferably. Electrophoresis in 1% agarose gel. Lanes: 1-8,pFsp4HI3/DriI, treated with serial 2-fold dilutions of ElmI (initial amount,added to the initial reaction mixture is 1 u.a.); 9, DNA molecular weightmarker 1 kb; 10-17, pFsp4HI3/DriI, treated with serial 2-fold dilutions of BisI(initial amount, added to the reaction mixture is 1 u.a.); 18, pFsp4HI3/DriI.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Incomplete cleavage of pFsp4HI3/DriI showing higher ElmI activity in5’-GCNGC-3’ sequences with three or four 5-methylcytosines,compared with 5’-GCNGC-3 sequences with only two 5-methylcytosines thatBisI cuts preferably. Electrophoresis in 1% agarose gel. Lanes: 1-8,pFsp4HI3/DriI, treated with serial 2-fold dilutions of ElmI (initial amount,added to the initial reaction mixture is 1 u.a.); 9, DNA molecular weightmarker 1 kb; 10-17, pFsp4HI3/DriI, treated with serial 2-fold dilutions of BisI(initial amount, added to the reaction mixture is 1 u.a.); 18, pFsp4HI3/DriI.
Mentions: In order to establish how effectively ElmI cleaves a 5’-GCNGC-3’sequence with a higher number of 5-methylcytosine residues, pFsp4HI3/DriI wastreated with ElmI in an amount insufficient for complete hydrolysis and theresult was compared with the result of its cleavage by BisI endonuclease(Figure 4).

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