Kinetics of Methylation by EcoP1I DNA Methyltransferase

Bheemanaik, Shivakumara; Sistla, Srivani; Krishnamurthy, Vinita; Arathi, Sampath; Desirazu, Narasimha Rao

doi:10.4061/2010/302731

Cited by 3 publications

(1 citation statement)

References 44 publications

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“…the P1 prophage-encoded type III M.EcoP1I. This MTase modifies ss- and dsDNA (similar K m and k cat ) with comparable efficiency and it was shown that ssDNA methylation is not linked with formation of double stranded structures 34 , 35 . Such unusual phage MTase feature may be related to the survival of single-stranded DNA phages in bacterial host 35 .…”

Section: Discussionmentioning

confidence: 99%

Isospecific adenine DNA methyltransferases show distinct preferences towards DNA substrates

Wons

Mruk

Kaczorowski

2018

Sci Rep

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Here, we report results on systematic analysis of DNA substrate preferences of three N6-adenine β-class DNA methyltransferases that are part of the type II restriction-modification systems. The studied enzymes were: M.EcoVIII, M.HindIII and M.LlaCI, which although found in phylogenetically distant bacteria (γ-proteobacteria and low-GC Gram-positive bacteria), recognize the same palindromic specific sequence 5′-AAGCTT-3′ and catalyze formation of N6-methyladenine at the first A-residue. As expected overall the enzymes share the most analyzed features, but they show also some distinct differences in substrate recognition. Therefore DNA methylation reactions were carried out not only under standard, but also under relaxed conditions using DMSO or glycerol. We found that all of these enzymes preferred DNA containing a hemimethylated target site, but differ in modification of ssDNA, especially more pronounced for M.EcoVIII under relaxed conditions. In these conditions they also have shown varied preferences toward secondary sites, which differ by one nucleotide from specific sequence. They preferred sequences with substitutions at the 1st (A1 → G/C) and at the 2nd position (A2 → C), while sites with substitutions at the 3rd position (G3 → A/C) were modified less efficiently. Kinetic parameters of the methylation reaction carried out by M.EcoVIII were determined. Methylation efficiency (kcat/Km) of secondary sites was 4.5–10 times lower when compared to the unmethylated specific sequences, whilst efficiency observed for the hemimethylated substrate was almost 4.5 times greater. We also observed a distinct effect of analyzed enzymes on unspecific interaction with DNA phosphate backbone. We concluded that for all three enzymes the most critical is the phosphodiester bond between G3-C4 nucleotides at the center of the target site.

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Section: Discussionmentioning

confidence: 99%

Isospecific adenine DNA methyltransferases show distinct preferences towards DNA substrates

Wons

Mruk

Kaczorowski

2018

Sci Rep

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Evaluation of the enzymatic properties of DNA (cytosine-5)-methyltransferase M.ApeKI from archaea in the presence of metal ions

Hayashi,

Wada,

Yamamura

et al. 2024

Bioscience, Biotechnology, and Biochemistry

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We previously identified M.ApeKI from Aeropyum pernix K1 as a highly thermostable DNA (cytosine-5)-methyltransferase. M.ApeKI uses the type II restriction-modification system (R-M system), among the best-studied R-M systems. Although endonucleases generally utilize Mg (II) as a cofactor, several reports have shown that MTases exhibit different reactions in the presence of metal ions. This study aim was to evaluate the enzymatic properties of DNA (cytosine-5)-methyltransferase M.ApeKI from archaea in the presence of metal ions. We evaluated the influence of metal ions on the catalytic activity and DNA binding of M.ApeKI. The catalytic activity was inhibited by Cu (II), Mg (II), Mn (II), and Zn (II), each at 5 mM. DNA binding was more strongly inhibited by 5 mM Cu (II) and 10 mM Zn (II). To our knowledge, this is the first report showing that DNA binding of typeII MTase is inhibited by metal ions.

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Type III restriction endonucleases are heterotrimeric: comprising one helicase–nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

Butterer

Pernstich²,

Smith³

et al. 2014

Nucleic Acids Research

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Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism.

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Kinetics of Methylation by EcoP1I DNA Methyltransferase

Cited by 3 publications

References 44 publications

Isospecific adenine DNA methyltransferases show distinct preferences towards DNA substrates

Isospecific adenine DNA methyltransferases show distinct preferences towards DNA substrates

Evaluation of the enzymatic properties of DNA (cytosine-5)-methyltransferase M.ApeKI from archaea in the presence of metal ions

Type III restriction endonucleases are heterotrimeric: comprising one helicase–nuclease subunit and a dimeric methyltransferase that binds only one specific DNA

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