Organization of restriction-modification systems

Wilson, Geoffrey G.

doi:10.1093/nar/19.10.2539

Cited by 230 publications

(160 citation statements)

References 149 publications

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“…To enable coexistence with their viruses, bacteria and archaea have evolved a variety of resistance mechanisms, including prevention of phage adsorption, the inhibition of DNA injection, restriction-modification systems and abortive infection systems [3][4][5][6] . Recent research, first described in 2002, has demonstrated the importance of a novel adaptive mechanism that provides bacteria immunity to phage 7,8 , that is based on clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated genes (cas) [9][10][11] .…”

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Strong bias in the bacterial CRISPR elements that confer immunity to phage

et al. 2013

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems provide adaptive immunity against phage via spacer-encoded CRISPR RNAs that are complementary to invasive nucleic acids. Here, we challenge Streptococcus thermophilus with a bacteriophage, and used PCR-based metagenomics to monitor phage-derived spacers daily for 15 days in two experiments. Spacers that target the host chromosome are infrequent and strongly selected against, suggesting autoimmunity is lethal. In experiments that recover over half a million spacers, we observe early dominance by a few spacer sub-populations and rapid oscillations in sub-population abundances. In two CRISPR systems and in replicate experiments, a few spacers account for the majority of spacer sequences. Nearly all phage locations targeted by the acquired spacers have a proto-spacer adjacent motif (PAM), indicating PAMs are involved in spacer acquisition. We detect a strong and reproducible bias in the phage genome locations from which spacers derive. This may reflect selection for specific spacers based on location and effectiveness.

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Strong bias in the bacterial CRISPR elements that confer immunity to phage

et al. 2013

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“…They catalyze the transfer of methyl groups from S-adenosyl-methionine (SAM) to specific nucleotides of double stranded DNA molecules. Methylases from type II RM systems (the most common) are encoded by separate proteins and act independently of their respective restriction endonucleases, whereas methylase and restriction activities of type I and III RM systems are provided by a unique protein complex (Wilson 1991).…”

Section: Methylasesmentioning

confidence: 99%

Enzymes used in molecular biology: a useful guide

Rittié

Perbal

2008

Journal of Cell Communication and Signaling

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Since molecular cloning has become routine laboratory technique, manufacturers offer countless sources of enzymes to generate and manipulate nucleic acids. Thus, selecting the appropriate enzyme for a specific task may seem difficult to the novice. This review aims at providing the readers with some cues for understanding the function and specificities of the different sources of polymerases, ligases, nucleases, phosphatases, methylases, and topoisomerases used for molecular cloning. We provide a description of the most commonly used enzymes of each group, and explain their properties and mechanism of action. By pointing out key requirements for each enzymatic activity and clarifying their limitations, we aim at guiding the reader in selecting appropriate enzymatic source and optimal experimental conditions for molecular cloning experiments.

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“…Understanding the molecular basis of sequence specificity requires the detailed structural and enzymological characterization of type-I1 DNA methyltransferases. The genes of several DNA methyltransferases have been cloned and sequenced [3], but relatively few enzymes have been purified to homogeneity and characterized enzymologically [6 -161.The EcaI DNA methyltransferase is part of the EcaI restriction-modification system that recognizes the sequence 5'-(GGTNACC)-3' [17, 181. We cloned the gene coding for Era1 DNA methyltransferase in Escherichia coli and determined its nucleotide sequence.…”

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“…The other product of the DNA-methylation reaction is S-adenosyl-L-homocysteine (AdoHcy). Most of the known sequence-specific DNA methyltransferases are part of restriction-modification systems, but there are enzymes which exist in the cell without a cognate restriction endonuclease [3]. Type-I1 DNA methyltransferases constitute an interesting example of sequence-specific DNA/protein interaction, especially if we consider the multitude of specificities discovered and the existence of different DNA methyltransferases recognizing the same sequence [4].…”

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Purification and biochemical characterization of the Ecal DNA methyltransferase

Szilák

Venetianer

Kiss

1992

European Journal of Biochemistry

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The EcaI GGTNACC-specific DNA-adenine modification methyltransferase has been purified to apparent homogeneity. The active form of the DNA methyltransferase is a single polypeptide. The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25°C. EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event. The K, was 170 nM for DNA and 1.8 pM for the methyl donor S-adenosylmethionine. Methylated DNA is a competitive inhibitor with respect to DNA (Ki = 3.5 nM). The other product of the DNA-methylation reaction, S-adenosylhomocysteine was found to be a competitive inhibitor with respect to S-adenosylmethionine (Ki = 2.7 pM). The Sadenosylmethionine analog sinefungin was shown to be a very strong inhibitor (Ki = 3.5 nM) of the DNA methyltransferase reaction.Type-I1 modification enzymes are sequence-specific DNA methyltransferases that recognize 4 -8-bp sequences in DNA and transfer one methyl group from the methyl donor Sadenosyl-L-methionine (AdoMet) to an adenine or cytosine of the recognition sequence in each strand [l]. These enzymes fall into three groups according to the methylated base generated: 5-methylcytosine, N4-methylcytosine and N6-methyladenine DNA methyltransferases [2]. The other product of the DNA-methylation reaction is S-adenosyl-L-homocysteine (AdoHcy). Most of the known sequence-specific DNA methyltransferases are part of restriction-modification systems, but there are enzymes which exist in the cell without a cognate restriction endonuclease [3]. Type-I1 DNA methyltransferases constitute an interesting example of sequence-specific DNA/protein interaction, especially if we consider the multitude of specificities discovered and the existence of different DNA methyltransferases recognizing the same sequence [4]. Type-I1 DNA methyltransferases have gained practical importance in the analysis of large genomes because of their use in increasing the specificity of certain restriction endonucleases [5]. Understanding the molecular basis of sequence specificity requires the detailed structural and enzymological characterization of type-I1 DNA methyltransferases. The genes of several DNA methyltransferases have been cloned and sequenced [3], but relatively few enzymes have been purified to homogeneity and characterized enzymologically [6 -161.The EcaI DNA methyltransferase is part of the EcaI restriction-modification system that recognizes the sequence 5'-(GGTNACC)-3' [17, 181. We cloned the gene coding for Era1 DNA methyltransferase in Escherichia coli and determined its nucleotide sequence. The nucleotide sequence of the ecaIM gene was used to derive the amino acid sequence of E d DNA methyltransferase [18]. EcaI DNA methyltransferase methylates the adenine of the 5'-GGTNACC-3' recognition sequence to yield p-methyladenine [18]. In this paper, we report the purification and initial enzymological characterization of EcaI DNA methyltransferase. MATERIALS AND METHODS Strains, plasmids and growth conditionsE. coli ER1398 [19] was used as ...

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Organization of restriction-modification systems

Cited by 230 publications

References 149 publications

Strong bias in the bacterial CRISPR elements that confer immunity to phage

Strong bias in the bacterial CRISPR elements that confer immunity to phage

Enzymes used in molecular biology: a useful guide

Purification and biochemical characterization of the Ecal DNA methyltransferase

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