Mutagenic scan of the H‐N‐H motif of colicin E9: implications for the mechanistic enzymology of colicins, homing enzymes and apoptotic endonucleases
Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H‐N‐H group of homing endonucleases. Crystal structures of the E9 DNase ha...
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Veröffentlicht in: | Nucleic acids research 2002-07, Vol.30 (14), p.3225-3234 |
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description | Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H‐N‐H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H‐N‐H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H‐N‐H motif is the site of DNA binding and that Mg2+‐dependent cleavage of double‐stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg2+ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation‐dependent catalytic mechanisms. The roles of individual amino acids within the H‐N‐H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase‐activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H‐N‐H DNases. |
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Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H‐N‐H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H‐N‐H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H‐N‐H motif is the site of DNA binding and that Mg2+‐dependent cleavage of double‐stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg2+ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation‐dependent catalytic mechanisms. The roles of individual amino acids within the H‐N‐H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase‐activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H‐N‐H DNases.</description><identifier>ISSN: 0305-1048</identifier><identifier>ISSN: 1362-4962</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gkf420</identifier><identifier>PMID: 12136104</identifier><identifier>CODEN: NARHAD</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Alanine - genetics ; Amino Acid Motifs - genetics ; Amino Acid Sequence ; Amino Acid Substitution ; Apoptosis ; Binding Sites - genetics ; Colicins - chemistry ; Colicins - genetics ; Colicins - metabolism ; Deoxyribonucleases - metabolism ; DNA - metabolism ; DNA-Binding Proteins - chemistry ; DNA-Binding Proteins - genetics ; DNA-Binding Proteins - metabolism ; Endonucleases - metabolism ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; Nickel - metabolism ; Protein Binding ; Protein Conformation ; Sequence Homology, Amino Acid ; Zinc - metabolism</subject><ispartof>Nucleic acids research, 2002-07, Vol.30 (14), p.3225-3234</ispartof><rights>Copyright Oxford University Press(England) Jul 15, 2002</rights><rights>Copyright © 2002 Oxford University Press 2002</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c469t-81231e3d4ed9c1872aaa50079a1b49657dd2d21b6694e088787d0bcd805adc043</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC135741/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC135741/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12136104$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Walker, David C.</creatorcontrib><creatorcontrib>Georgiou, Theonie</creatorcontrib><creatorcontrib>Pommer, Ansgar J.</creatorcontrib><creatorcontrib>Walker, Daniel</creatorcontrib><creatorcontrib>Moore, Geoffrey R.</creatorcontrib><creatorcontrib>Kleanthous, Colin</creatorcontrib><creatorcontrib>James, Richard</creatorcontrib><title>Mutagenic scan of the H‐N‐H motif of colicin E9: implications for the mechanistic enzymology of colicins, homing enzymes and apoptotic endonucleases</title><title>Nucleic acids research</title><addtitle>Nucl. Acids Res</addtitle><description>Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H‐N‐H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H‐N‐H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H‐N‐H motif is the site of DNA binding and that Mg2+‐dependent cleavage of double‐stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg2+ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation‐dependent catalytic mechanisms. The roles of individual amino acids within the H‐N‐H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase‐activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H‐N‐H DNases.</description><subject>Alanine - genetics</subject><subject>Amino Acid Motifs - genetics</subject><subject>Amino Acid Sequence</subject><subject>Amino Acid Substitution</subject><subject>Apoptosis</subject><subject>Binding Sites - genetics</subject><subject>Colicins - chemistry</subject><subject>Colicins - genetics</subject><subject>Colicins - metabolism</subject><subject>Deoxyribonucleases - metabolism</subject><subject>DNA - metabolism</subject><subject>DNA-Binding Proteins - chemistry</subject><subject>DNA-Binding Proteins - genetics</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>Endonucleases - metabolism</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutation</subject><subject>Nickel - metabolism</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Sequence Homology, Amino Acid</subject><subject>Zinc - metabolism</subject><issn>0305-1048</issn><issn>1362-4962</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpdkc1u1DAUhS0EotPChgdAFgsWFaHXseMklVigqmWQWv7En9hYHtvJuE3sYCeIYcUjsOT5eBJcMiqFhWX5nu9c3euD0D0CjwnU9MDJcNBeNCyHG2hBKM8zVvP8JloAhSIjwKodtBvjOQBhpGC30Q7JE5aEBfp5No2yNc4qHJV02Dd4XBu8_PX9x4t0lrj3o20uy8p3VlmHj-tDbPshPeRovYu48eGPpzdqLZ2NY-pl3LdN7zvfbq5Z4yO89r117SybiKXTWA5-GP1s0t5NqjMymngH3WpkF83d7b2H3p0cvz1aZqcvnz0_enqaKcbrMatITomhmhldK1KVuZSyAChrSVbpF4pS61znZMV5zQxUVVmVGlZKV1BIrYDRPfRk7jtMq95oZdwYZCeGYHsZNsJLK_5VnF2L1n8RhBYlI8n_cOsP_vNk4ih6G5XpOumMn6IgFeMVQJHAB_-B534KLu0mcgDOaV3UCdqfIRV8jME0V4MQEJdhixS2mMNO8P3ro_9Ft-kmIJuBFIr5eqXLcCF4SctCLD9-Eq8-nL0hr0-4eE9_A2Wxujw</recordid><startdate>20020715</startdate><enddate>20020715</enddate><creator>Walker, David C.</creator><creator>Georgiou, Theonie</creator><creator>Pommer, Ansgar J.</creator><creator>Walker, Daniel</creator><creator>Moore, Geoffrey R.</creator><creator>Kleanthous, Colin</creator><creator>James, Richard</creator><general>Oxford University Press</general><general>Oxford Publishing Limited (England)</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7QP</scope><scope>7QR</scope><scope>7SS</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>5PM</scope></search><sort><creationdate>20020715</creationdate><title>Mutagenic scan of the H‐N‐H motif of colicin E9: implications for the mechanistic enzymology of colicins, homing enzymes and apoptotic endonucleases</title><author>Walker, David C. ; Georgiou, Theonie ; Pommer, Ansgar J. ; Walker, Daniel ; Moore, Geoffrey R. ; Kleanthous, Colin ; James, Richard</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c469t-81231e3d4ed9c1872aaa50079a1b49657dd2d21b6694e088787d0bcd805adc043</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Alanine - genetics</topic><topic>Amino Acid Motifs - genetics</topic><topic>Amino Acid Sequence</topic><topic>Amino Acid Substitution</topic><topic>Apoptosis</topic><topic>Binding Sites - genetics</topic><topic>Colicins - chemistry</topic><topic>Colicins - genetics</topic><topic>Colicins - metabolism</topic><topic>Deoxyribonucleases - metabolism</topic><topic>DNA - metabolism</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>DNA-Binding Proteins - genetics</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>Endonucleases - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Mutation</topic><topic>Nickel - metabolism</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Sequence Homology, Amino Acid</topic><topic>Zinc - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Walker, David C.</creatorcontrib><creatorcontrib>Georgiou, Theonie</creatorcontrib><creatorcontrib>Pommer, Ansgar J.</creatorcontrib><creatorcontrib>Walker, Daniel</creatorcontrib><creatorcontrib>Moore, Geoffrey R.</creatorcontrib><creatorcontrib>Kleanthous, Colin</creatorcontrib><creatorcontrib>James, Richard</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Walker, David C.</au><au>Georgiou, Theonie</au><au>Pommer, Ansgar J.</au><au>Walker, Daniel</au><au>Moore, Geoffrey R.</au><au>Kleanthous, Colin</au><au>James, Richard</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mutagenic scan of the H‐N‐H motif of colicin E9: implications for the mechanistic enzymology of colicins, homing enzymes and apoptotic endonucleases</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucl. Acids Res</addtitle><date>2002-07-15</date><risdate>2002</risdate><volume>30</volume><issue>14</issue><spage>3225</spage><epage>3234</epage><pages>3225-3234</pages><issn>0305-1048</issn><issn>1362-4962</issn><eissn>1362-4962</eissn><coden>NARHAD</coden><abstract>Colicin E9 is a microbial toxin that kills bacteria through random degradation of chromosomal DNA. Within the active site of the cytotoxic endonuclease domain of colicin E9 (the E9 DNase) is a 32 amino acid motif found in the H‐N‐H group of homing endonucleases. Crystal structures of the E9 DNase have implicated several conserved residues of the H‐N‐H motif in the mechanism of DNA hydrolysis. We have used mutagenesis to test the involvement of these key residues in colicin toxicity, metal ion binding and catalysis. Our data show, for the first time, that the H‐N‐H motif is the site of DNA binding and that Mg2+‐dependent cleavage of double‐stranded DNA is responsible for bacterial cell death. We demonstrate that more active site residues are required for catalysis in the presence of Mg2+ ions than transition metals, consistent with the recent hypothesis that the E9 DNase hydrolyses DNA by two distinct, cation‐dependent catalytic mechanisms. The roles of individual amino acids within the H‐N‐H motif are discussed in the context of the available structural information on this and related DNases and we address the possible mechanistic similarities between caspase‐activated DNases, responsible for the degradation of chromatin in eukaryotic apoptosis, and H‐N‐H DNases.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>12136104</pmid><doi>10.1093/nar/gkf420</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Alanine - genetics Amino Acid Motifs - genetics Amino Acid Sequence Amino Acid Substitution Apoptosis Binding Sites - genetics Colicins - chemistry Colicins - genetics Colicins - metabolism Deoxyribonucleases - metabolism DNA - metabolism DNA-Binding Proteins - chemistry DNA-Binding Proteins - genetics DNA-Binding Proteins - metabolism Endonucleases - metabolism Molecular Sequence Data Mutagenesis, Site-Directed Mutation Nickel - metabolism Protein Binding Protein Conformation Sequence Homology, Amino Acid Zinc - metabolism |
title | Mutagenic scan of the H‐N‐H motif of colicin E9: implications for the mechanistic enzymology of colicins, homing enzymes and apoptotic endonucleases |
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