Isolation and Characterization of Site-Specific DNA-methyltransferases from Bacillus coagulans K

Svad’bina, I. V.; Zelinskaya, Natalia; Kovalevskaya, N. P.; Zheleznaya, L. A.; Matvienko, N. I.

doi:10.1023/b:biry.0000022061.29918.8b

Cited by 4 publications

(3 citation statements)

References 8 publications

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“…Next, the 14 C-methylated oligoduplex was hydrolyzed in 10 l of 60% perchloric acid at 95°C for 1 h (31), followed by the addition of 20 l of 1 M KOH to neutralize the reaction mixture. The resulting insoluble KClO 4 was removed by centrifugation (56), and the 14 Cmethylated bases in the supernatant were separated on a DC-Platten cellulose plate (EMD Biosciences, Inc., Merck, San Diego, CA) with a liquid phase of isopropanol-water-NH 4 OH (60:10:0.1 [vol/vol/vol]) at 25°C for 6 h. The 14 Cmethylated bases on the air-dried filters were exposed to a BAS-MS2025 imaging plate and analyzed using an FLA-5100 imaging system.…”

Section: Methodsmentioning

confidence: 99%

Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

Watanabe

Yuzawa

Handa

et al. 2006

Appl Environ Microbiol

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Genome sequence comparisons among multiple species of Pyrococcus, a hyperthermophilic archaeon, revealed a linkage between a putative restriction-modification gene complex and several large genome polymorphisms/rearrangements. From a region apparently inserted into the Pyrococcus abyssi genome, a hyperthermoresistant restriction enzyme [PabI; 5-(GTA/C)] with a novel structure was discovered. In the present work, the neighboring methyltransferase homologue, M.PabI, was characterized. Its N-terminal half showed high similarities to the M subunit of type I systems and a modification enzyme of an atypical type II system, M.AhdI, while its C-terminal half showed high similarity to the S subunit of type I systems. M.PabI expressed within Escherichia coli protected PabI sites from RsaI, a PabI isoschizomer. M.PabI, purified following overexpression, was shown to generate 5-GTm6AC, which provides protection against PabI digestion. M.PabI was found to be highly thermophilic; it showed methylation at 95°C and retained at least half the activity after 9 min at 95°C. This hyperthermophilicity allowed us to obtain activation energy and other thermodynamic parameters for the first time for any DNA methyltransferases. We also determined the kinetic parameters of k cat , K m, DNA , and K m, AdoMet . The activity of M.PabI was optimal at a slightly acidic pH and at an NaCl concentration of 200 to 500 mM and was inhibited by Zn 2؉ but not by Mg 2؉ , Ca 2؉ , or Mn 2؉ . These and previous results suggest that this unique methyltransferase and PabI constitute a type II restriction-modification gene complex that inserted into the P. abyssi genome relatively recently. As the most thermophilic of all the characterized DNA methyltransferases, M.PabI may help in the analysis of DNA methylation and its application to DNA engineering.DNA methylation plays a crucial role in diverse biological processes, for example, replication and repair of DNA, expression and silencing of genes, and distinction between self and nonself DNAs. DNA methyltransferases require S-adenosyl-Lmethionine (AdoMet) as the donor of a methyl group (7). The common core of the AdoMet-dependent methyltransferases is formed by seven-stranded ␤-sheets (53), and the target base within a double-stranded DNA molecule is installed into its catalytic pocket by a "base-flipping" mechanism (20). The AdoMet-dependent DNA methyltransferases are classified into three groups according to their products: those yielding 5-methylcytosine (m5C), those yielding N 4 -methylcytosine (m4C), and those yielding N 6 -methyladenine (m6A). The latter two groups of amino-nitrogen methyltransferases are further subdivided into ␣, ␤, ␥, and other families, according to the order of motifs (of the catalytic center and AdoMet-binding region) and the target recognition domain (TRD) (4, 27).Most of the prokaryotic DNA methyltransferases constitute restriction-modification (RM) systems, which can be classified into types I, II, and III (45,46). A classic type II RM system is composed of two distinct prote...

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Section: Methodsmentioning

confidence: 99%

Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

Watanabe

Yuzawa

Handa

et al. 2006

Appl Environ Microbiol

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“…M.BcoKIA modifies cytosine in the recognition sequence, and M.BcoKIB modifies adenine at the N4 position [6]. Here we report the location of the bases modified by these methylases in the recognition sequence.…”

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confidence: 71%

Location of the Bases Modified by M.BcoKIA and M.BcoKIB Methylases in the Sequence 5′-CTCTTC-3′/5′-GAAGAG-3′

Svad’bina

Zheleznaya

Matvienko

2005

Biochemistry (Moscow)

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The strain Bacillus coagulans K contains two DNA-methyltransferases, M.BcoKIA and M.BcoKIB, which recognize the sequence 5 -CTCTTC-3 /5 -GAAGAG-3 and possess N4-methylcytosine and N6-methyladenine specificities, respectively. A special construct containing the recognition site of BcoKI and sites of four IIS restriction endonucleases (IIS restriction endonuclease cassette) was designed to locate the nucleotides modified by the methylases. The modified bases were determined as: 5 -m(4)CTCTTC-3 /5 -GAAGAm(6)G-3 .

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“…At present, a variety of methods have been proposed to detect the 6mA events in eukaryotic and prokaryotic genomes, including bisulfite sequencing (Svadbina et al, 2004), methylated DNA immunoprecipitation sequencing (MeDIP-seq) (Zhao et al, 2014), restriction enzyme-based 6mA sequencing (REseq) (Luo et al, 2016), single-molecule real-time sequencing (SMRT-seq) (Flusberg et al, 2010) and Nanopore sequencing (ONT-seq) (Branton et al, 2008). Previously, the whole genome DNA methylation detection mainly relied on bisulfite sequencing or the next generation sequencing of methylated DNA immunoprecipitation (Shanmuganathan et al, 2013), but it was difficult to accurately identify the methylation of genomic repeat regions due to the short reads.…”

Section: Introductionmentioning

confidence: 99%

MASQC: Next Generation Sequencing Assists Third Generation Sequencing for Quality Control in N6-Methyladenine DNA Identification

et al. 2020

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DNA N6-methyladenine (6mA) modification has been discovered as the most prevalent DNA modification in prokaryotes and eukaryotes, involving gene expression, DNA replication and repair, and host-pathogen interactions. Single-molecule real-time sequencing (SMRT-seq) can detect 6mA events in prokaryotic and eukaryotic genomes at the single-nucleotide level. However, there are no strict and economical quality control methods for high false-positive 6mA events in eukaryotic genomes. Therefore, by analyzing the distribution of 6mA in eukaryotic and prokaryotes, we proposed a method named MASQC (MeDIP-seq assists SMRT-seq for quality control in 6mA identification), which can identify 6mA events without doing the whole genome amplification (WGA) sequencing. The proposed MASQC method was assessed on two eukaryotic genomes and six bacterial genomes, our results demonstrate that MASQC performs well in quality control of false positive 6mA identification for both eukaryotic and prokaryotic genomes.

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Isolation and Characterization of Site-Specific DNA-methyltransferases from Bacillus coagulans K

Cited by 4 publications

References 8 publications

Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

Hyperthermophilic DNA Methyltransferase M.PabI from the Archaeon Pyrococcus abyssi

Location of the Bases Modified by M.BcoKIA and M.BcoKIB Methylases in the Sequence 5′-CTCTTC-3′/5′-GAAGAG-3′

MASQC: Next Generation Sequencing Assists Third Generation Sequencing for Quality Control in N6-Methyladenine DNA Identification

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