Direct and continuous fluorescence-based measurements of Pyrococcus horikoshii DNA N-6 adenine methyltransferase activity

Maynard-Smith, Michael D.; McKelvie, Jennifer C.; Wood, R.J.K.; Harmer, Jenny E.; Ranasinghe, Rohan T.; Williams, Claire L; Coomber, Danielle M.; Stares, Alexandra F.; Roach, Peter L.

doi:10.1016/j.ab.2011.07.023

Cited by 3 publications

(3 citation statements)

References 42 publications

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“…Whether changes in curvature, especially in regions of high GATC content such as oriC , promote binding of proteins, such as SeqA, or play a role in origin function is not known. The destabilization of a methylated 13-bp duplex oligonucleotide is the basis for a fluorescence-based assay for Dam methyltransferase activity [32]. …”

Section: Methylated Bases In Dnamentioning

confidence: 99%

DNA Methylation

Marinus

Løbner-Olesen

2014

EcoSal Plus

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The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function.

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Section: Methylated Bases In Dnamentioning

confidence: 99%

DNA Methylation

Marinus

Løbner-Olesen

2014

EcoSal Plus

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“…Widely prevalent across bacteria, some archaea also harbor the Dam. Halobacterium saccharovorum and Methanobacterium strain Ivanov have been reported to be Dam + as early as 1984 ( Barbeyron et al, 1984 ), and the in vitro methylation activity of the Dam methylase of Pyrococcus horikoshii has also been examined ( Maynard-Smith et al, 2011 ). Koike et al (2005) identified the Dam methylase in multiple archaea species through annotation of genome sequences.…”

Section: Discussionmentioning

confidence: 99%

The extremophile Picrophilus torridus carries a DNA adenine methylase M.PtoI that is part of a Type I restriction-modification system

et al. 2023

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DNA methylation events mediated by orphan methyltransferases modulate various cellular processes like replication, repair and transcription. Bacteria and archaea also harbor DNA methyltransferases that are part of restriction-modification systems, which serve to protect the host genome from being cleaved by the cognate restriction enzyme. While DNA methylation has been exhaustively investigated in bacteria it remains poorly understood in archaea. Picrophilus torridus is a euryarchaeon that can thrive under conditions of extremely low pH (0.7), and thus far no reports have been published regarding DNA methylation in this extremophile. This study reports the first experimentation examining DNA methylation in P. torridus. We find the genome to carry methylated adenine (m6A) but not methylated cytosine (m5C) residues. The m6A modification is absent at GATC sites, indicating the absence of an active Dam methylase even though the dam gene has been annotated in the genome sequence. Two other methylases have also been annotated in the P. torridus genome sequence. One of these is a part of a Type I restriction-modification system. Considering that all Type I modification methylases characterized to date target adenine residues, the modification methylase of this Type I system has been examined. The genes encoding the S subunit (that is responsible for DNA recognition) and M subunit (that is responsible for DNA methylation) have been cloned and the recombinant protein purified from E.coli, and regions involved in M-S interactions have been identified. The M.PtoI enzyme harbors all the motifs that typify Type I modification methylases, and displays robust adenine methylation in in vitro assays under a variety of conditions. Interestingly, magnesium is essential for enzyme activity. The enzyme displays substrate inhibition at higher concentrations of AdoMet. Mutational analyses reveal that Motif I plays a role in AdoMet binding, and Motif IV is critical for methylation activity. The data presented here lays the foundation for further research in the area of DNA methylation and restriction-modification research in this most unusual microorganism.

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“…Therefore, MTases have an essential role in life evolution [ 1 ], especially in the cell cycle and regulation of gene transcription [ 63 ]. Several examples of archaea MTases have been studied; there are Haloferax volcanii [ 64 ], Pyrocuccus abyssi [ 65 ], Pyrococcus horikoshii [ 66 , 67 ], Sulfolobus solfactaricus [ 68 ], and Thermococcus gammatolerans [ 69 ]. In bacteria, examples include Alphaproteobacteria [ 36 ], Borrelia burgdorferi [ 70 ], Escherichia coli [ 71 ], Helicobacter pylori [ 72 ], and also Mycobacterium species [ 73 ].…”

Section: Dna Methyltransferasesmentioning

confidence: 99%

DNA Methyltransferases: From Evolution to Clinical Applications

Falconi

Torres-Arciga

Matus-Ortega

et al. 2022

IJMS

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DNA methylation is an epigenetic mark that living beings have used in different environments. The MTases family catalyzes DNA methylation. This process is conserved from archaea to eukaryotes, from fertilization to every stage of development, and from the early stages of cancer to metastasis. The family of DNMTs has been classified into DNMT1, DNMT2, and DNMT3. Each DNMT has been duplicated or deleted, having consequences on DNMT structure and cellular function, resulting in a conserved evolutionary reaction of DNA methylation. DNMTs are conserved in the five kingdoms of life: bacteria, protists, fungi, plants, and animals. The importance of DNMTs in whether methylate or not has a historical adaptation that in mammals has been discovered in complex regulatory mechanisms to develop another padlock to genomic insurance stability. The regulatory mechanisms that control DNMTs expression are involved in a diversity of cell phenotypes and are associated with pathologies transcription deregulation. This work focused on DNA methyltransferases, their biology, functions, and new inhibitory mechanisms reported. We also discuss different approaches to inhibit DNMTs, the use of non-coding RNAs and nucleoside chemical compounds in recent studies, and their importance in biological, clinical, and industry research.

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Direct and continuous fluorescence-based measurements of Pyrococcus horikoshii DNA N-6 adenine methyltransferase activity

Cited by 3 publications

References 42 publications

DNA Methylation

DNA Methylation

The extremophile Picrophilus torridus carries a DNA adenine methylase M.PtoI that is part of a Type I restriction-modification system

DNA Methyltransferases: From Evolution to Clinical Applications

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