DNA Recognition by the EcoK Methyltransferase : The Influence of DNA Methylation and the Cofactor S-adenosyl- l-methionine
The methyltransferase of the EcoK type I restriction/modification system is trimeric, M 2S 1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the m...
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Veröffentlicht in: | Journal of molecular biology 1993-11, Vol.234 (1), p.60-71 |
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description | The methyltransferase of the
EcoK type I restriction/modification system is trimeric, M
2S
1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor
S -adenosyl-
l-methionine. Our results indicate that the methyltransferase has two non-interacting
S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3·60(± 0·42) μM determined by equilibrium dialysis, or 2·21(±0·29) μM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that
S -adenosyl-
l-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to
S -adenosyl-
l-methionine binding. Gel retardation results show that the binding of
S-adenosyl-
l-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)
6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis. |
doi_str_mv | 10.1006/jmbi.1993.1563 |
format | Article |
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EcoK type I restriction/modification system is trimeric, M
2S
1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor
S -adenosyl-
l-methionine. Our results indicate that the methyltransferase has two non-interacting
S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3·60(± 0·42) μM determined by equilibrium dialysis, or 2·21(±0·29) μM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that
S -adenosyl-
l-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to
S -adenosyl-
l-methionine binding. Gel retardation results show that the binding of
S-adenosyl-
l-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)
6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.</description><identifier>ISSN: 0022-2836</identifier><identifier>EISSN: 1089-8638</identifier><identifier>DOI: 10.1006/jmbi.1993.1563</identifier><identifier>PMID: 8230207</identifier><identifier>CODEN: JMOBAK</identifier><language>eng</language><publisher>Oxford: Elsevier Ltd</publisher><subject>Allosteric Regulation ; Analytical, structural and metabolic biochemistry ; Bacterial Proteins - metabolism ; Base Sequence ; Biological and medical sciences ; DNA methylation ; DNA-Binding Proteins - metabolism ; DNA-protein interactions ; Enzymes and enzyme inhibitors ; Escherichia coli ; Fundamental and applied biological sciences. Psychology ; Hydrolases ; In Vitro Techniques ; Kinetics ; Methylation ; methyltransferase ; Molecular Sequence Data ; Oligodeoxyribonucleotides - metabolism ; Osmolar Concentration ; Protein Binding ; Protein Conformation ; restriction and modification ; S-adenosyl- l-methionine ; S-Adenosylmethionine - metabolism ; Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism ; Substrate Specificity</subject><ispartof>Journal of molecular biology, 1993-11, Vol.234 (1), p.60-71</ispartof><rights>1993 Academic Press</rights><rights>1994 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1006/jmbi.1993.1563$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=3809332$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/8230207$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Powell, Lynn M.</creatorcontrib><creatorcontrib>Dryden, David T.F.</creatorcontrib><creatorcontrib>Willcock, Damion F.</creatorcontrib><creatorcontrib>Pain, Roger H.</creatorcontrib><creatorcontrib>Murray, Noreen E.</creatorcontrib><title>DNA Recognition by the EcoK Methyltransferase : The Influence of DNA Methylation and the Cofactor S-adenosyl- l-methionine</title><title>Journal of molecular biology</title><addtitle>J Mol Biol</addtitle><description>The methyltransferase of the
EcoK type I restriction/modification system is trimeric, M
2S
1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor
S -adenosyl-
l-methionine. Our results indicate that the methyltransferase has two non-interacting
S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3·60(± 0·42) μM determined by equilibrium dialysis, or 2·21(±0·29) μM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that
S -adenosyl-
l-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to
S -adenosyl-
l-methionine binding. Gel retardation results show that the binding of
S-adenosyl-
l-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)
6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.</description><subject>Allosteric Regulation</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Bacterial Proteins - metabolism</subject><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>DNA methylation</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>DNA-protein interactions</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Escherichia coli</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hydrolases</subject><subject>In Vitro Techniques</subject><subject>Kinetics</subject><subject>Methylation</subject><subject>methyltransferase</subject><subject>Molecular Sequence Data</subject><subject>Oligodeoxyribonucleotides - metabolism</subject><subject>Osmolar Concentration</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>restriction and modification</subject><subject>S-adenosyl- l-methionine</subject><subject>S-Adenosylmethionine - metabolism</subject><subject>Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism</subject><subject>Substrate Specificity</subject><issn>0022-2836</issn><issn>1089-8638</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v1DAURS1UVKaFLTskL6ruMjzHE8dmV00_1QISlLXl2M_UVWK3cQZp-PU4nRHbrt7iHh3p3UvIRwZLBiA-Pw5dWDKl-JI1gr8hCwZSVVJweUAWAHVd1ZKLd-Qo50cAaPhKHpJDWXOooV2Qv-ffzugPtOl3DFNIkXZbOj0gvbDpln7F6WHbT6OJ2eNoMtIv9L6EN9H3G4wWafJ0FuxA8yIw0b0Y1skbO6WR_qyMw5jytq9oXw0FLViI-J689abP-GF_j8mvy4v79XV19_3qZn12VyFnzVR5q8wKDfJOeqEMgjLMONla1TAn3Ao6zqEBj6J1aLlVq1Z4Z30rlarBO35MTnfepzE9bzBPegjZYt-biGmTdSugEaWXV0EmJBNNywr4aQ9uugGdfhrDYMat3rda8pN9brI1vS8F2pD_Y1yC4rwumNxhWL7_E3DU2Ya5VhdGtJN2KWgGep5ZzzPreWY9z8z_AVsTmbM</recordid><startdate>19931105</startdate><enddate>19931105</enddate><creator>Powell, Lynn M.</creator><creator>Dryden, David T.F.</creator><creator>Willcock, Damion F.</creator><creator>Pain, Roger H.</creator><creator>Murray, Noreen E.</creator><general>Elsevier Ltd</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7QL</scope><scope>7TM</scope><scope>C1K</scope><scope>7X8</scope></search><sort><creationdate>19931105</creationdate><title>DNA Recognition by the EcoK Methyltransferase : The Influence of DNA Methylation and the Cofactor S-adenosyl- l-methionine</title><author>Powell, Lynn M. ; Dryden, David T.F. ; Willcock, Damion F. ; Pain, Roger H. ; Murray, Noreen E.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-e315t-fc9a4eae3b8f69ae09a1ad87c951d6d40b33050fe67dec3c9476fdcf789920fd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>Allosteric Regulation</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Bacterial Proteins - metabolism</topic><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>DNA methylation</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>DNA-protein interactions</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Escherichia coli</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hydrolases</topic><topic>In Vitro Techniques</topic><topic>Kinetics</topic><topic>Methylation</topic><topic>methyltransferase</topic><topic>Molecular Sequence Data</topic><topic>Oligodeoxyribonucleotides - metabolism</topic><topic>Osmolar Concentration</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>restriction and modification</topic><topic>S-adenosyl- l-methionine</topic><topic>S-Adenosylmethionine - metabolism</topic><topic>Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Powell, Lynn M.</creatorcontrib><creatorcontrib>Dryden, David T.F.</creatorcontrib><creatorcontrib>Willcock, Damion F.</creatorcontrib><creatorcontrib>Pain, Roger H.</creatorcontrib><creatorcontrib>Murray, Noreen E.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Nucleic Acids Abstracts</collection><collection>Environmental Sciences and Pollution Management</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of molecular biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Powell, Lynn M.</au><au>Dryden, David T.F.</au><au>Willcock, Damion F.</au><au>Pain, Roger H.</au><au>Murray, Noreen E.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>DNA Recognition by the EcoK Methyltransferase : The Influence of DNA Methylation and the Cofactor S-adenosyl- l-methionine</atitle><jtitle>Journal of molecular biology</jtitle><addtitle>J Mol Biol</addtitle><date>1993-11-05</date><risdate>1993</risdate><volume>234</volume><issue>1</issue><spage>60</spage><epage>71</epage><pages>60-71</pages><issn>0022-2836</issn><eissn>1089-8638</eissn><coden>JMOBAK</coden><abstract>The methyltransferase of the
EcoK type I restriction/modification system is trimeric, M
2S
1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor
S -adenosyl-
l-methionine. Our results indicate that the methyltransferase has two non-interacting
S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3·60(± 0·42) μM determined by equilibrium dialysis, or 2·21(±0·29) μM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that
S -adenosyl-
l-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to
S -adenosyl-
l-methionine binding. Gel retardation results show that the binding of
S-adenosyl-
l-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)
6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.</abstract><cop>Oxford</cop><pub>Elsevier Ltd</pub><pmid>8230207</pmid><doi>10.1006/jmbi.1993.1563</doi><tpages>12</tpages></addata></record> |
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source | MEDLINE; Access via ScienceDirect (Elsevier) |
subjects | Allosteric Regulation Analytical, structural and metabolic biochemistry Bacterial Proteins - metabolism Base Sequence Biological and medical sciences DNA methylation DNA-Binding Proteins - metabolism DNA-protein interactions Enzymes and enzyme inhibitors Escherichia coli Fundamental and applied biological sciences. Psychology Hydrolases In Vitro Techniques Kinetics Methylation methyltransferase Molecular Sequence Data Oligodeoxyribonucleotides - metabolism Osmolar Concentration Protein Binding Protein Conformation restriction and modification S-adenosyl- l-methionine S-Adenosylmethionine - metabolism Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism Substrate Specificity |
title | DNA Recognition by the EcoK Methyltransferase : The Influence of DNA Methylation and the Cofactor S-adenosyl- l-methionine |
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