Determinants of EcoRI Endonuclease Sequence Discrimination
The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases w...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1989-05, Vol.86 (10), p.3579-3583 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Needels, M. C. Fried, S. R. Love, R. Rosenberg, J. M. Boyer, H. W. Greene, P. J. |
| description | The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro. The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity. Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp). The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200. The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase. In crude cell lysates, only the Cys mutant shows a very low level of in vitro activity. None of the mutant enzymes show a preference for alternative sites in assays in vitro. The implications of these results are discussed. |
| doi_str_mv | 10.1073/pnas.86.10.3579 |
| format | Article |
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C. ; Fried, S. R. ; Love, R. ; Rosenberg, J. M. ; Boyer, H. W. ; Greene, P. J.</creator><creatorcontrib>Needels, M. C. ; Fried, S. R. ; Love, R. ; Rosenberg, J. M. ; Boyer, H. W. ; Greene, P. J.</creatorcontrib><description>The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro. The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity. Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp). The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200. The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase. In crude cell lysates, only the Cys mutant shows a very low level of in vitro activity. None of the mutant enzymes show a preference for alternative sites in assays in vitro. The implications of these results are discussed.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.86.10.3579</identifier><identifier>PMID: 2657723</identifier><identifier>CODEN: PNASA6</identifier><language>eng</language><publisher>Washington, DC: National Academy of Sciences of the United States of America</publisher><subject>Amino acids ; Analytical, structural and metabolic biochemistry ; Arginine ; Bacterial Proteins - ultrastructure ; Binding Sites ; Biological and medical sciences ; Cell culture techniques ; Curves ; Deoxyribonuclease EcoRI - genetics ; Deoxyribonuclease EcoRI - metabolism ; DNA ; DNA cleavage ; DNA Mutational Analysis ; DNA-Binding Proteins - metabolism ; DNA-Binding Proteins - ultrastructure ; Enzymes ; Enzymes and enzyme inhibitors ; Escherichia coli - enzymology ; Escherichia coli - growth & development ; Fundamental and applied biological sciences. Psychology ; Hydrogen Bonding ; Hydrogen bonds ; Hydrolases ; Models, Molecular ; Phenotypes ; Plasmids ; Protein Conformation ; Structure-Activity Relationship ; Substrate Specificity ; Viability</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1989-05, Vol.86 (10), p.3579-3583</ispartof><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c521t-7f4d139252135c49c56bb4fb8b5e8ae2af249af2845d0f0d37501aa688ffe5563</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/86/10.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/33916$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/33916$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,727,780,784,803,885,27923,27924,53790,53792,58016,58249</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6968956$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2657723$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Needels, M. C.</creatorcontrib><creatorcontrib>Fried, S. R.</creatorcontrib><creatorcontrib>Love, R.</creatorcontrib><creatorcontrib>Rosenberg, J. M.</creatorcontrib><creatorcontrib>Boyer, H. W.</creatorcontrib><creatorcontrib>Greene, P. J.</creatorcontrib><title>Determinants of EcoRI Endonuclease Sequence Discrimination</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro. The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity. Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp). The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200. The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase. In crude cell lysates, only the Cys mutant shows a very low level of in vitro activity. None of the mutant enzymes show a preference for alternative sites in assays in vitro. The implications of these results are discussed.</description><subject>Amino acids</subject><subject>Analytical, structural and metabolic biochemistry</subject><subject>Arginine</subject><subject>Bacterial Proteins - ultrastructure</subject><subject>Binding Sites</subject><subject>Biological and medical sciences</subject><subject>Cell culture techniques</subject><subject>Curves</subject><subject>Deoxyribonuclease EcoRI - genetics</subject><subject>Deoxyribonuclease EcoRI - metabolism</subject><subject>DNA</subject><subject>DNA cleavage</subject><subject>DNA Mutational Analysis</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>DNA-Binding Proteins - ultrastructure</subject><subject>Enzymes</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - growth & development</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Hydrogen Bonding</subject><subject>Hydrogen bonds</subject><subject>Hydrolases</subject><subject>Models, Molecular</subject><subject>Phenotypes</subject><subject>Plasmids</subject><subject>Protein Conformation</subject><subject>Structure-Activity Relationship</subject><subject>Substrate Specificity</subject><subject>Viability</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1989</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkctrFTEUxoMo9VpdC4IyC7Gruc37IbiQ9lYLBcHHOmQyiU6Zm9wmGdH_3kzv9Kob3SQcvt85-U4-AJ4iuEZQkNNdMHkteS3WhAl1D6wQVKjlVMH7YAUhFq2kmD4Ej3K-hhAqJuEROMKcCYHJCrw-d8Wl7RBMKLmJvtnY-PGy2YQ-hsmOzmTXfHI3kwvWNedDtmmY4TLE8Bg88GbM7slyH4MvF5vPZ-_bqw_vLs_eXrWWYVRa4WmPiMK1IMxSZRnvOuo72TEnjcPGY6rqISnroYc9EQwiY7iU3jvGODkGb_Zzd1O3db11oSQz6l11YtJPHc2g_1bC8E1_jd81lgJJVPtfLf0p1kVy0du6hxtHE1ycshZSKQEZ_i-IGJMUytnR6R60KeacnD-YQVDPseg5Fi35XM-x1I7nf-5w4Jccqv5y0U22ZvTJBDvkA8YVl-r2K04WbJ5_p_5-R_tpHIv7USr54p9kBZ7tgetcYjoQhCjEyS_JerdB</recordid><startdate>19890501</startdate><enddate>19890501</enddate><creator>Needels, M. C.</creator><creator>Fried, S. R.</creator><creator>Love, R.</creator><creator>Rosenberg, J. M.</creator><creator>Boyer, H. W.</creator><creator>Greene, P. J.</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><scope>IQODW</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>7TM</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19890501</creationdate><title>Determinants of EcoRI Endonuclease Sequence Discrimination</title><author>Needels, M. C. ; Fried, S. R. ; Love, R. ; Rosenberg, J. M. ; Boyer, H. W. ; Greene, P. J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c521t-7f4d139252135c49c56bb4fb8b5e8ae2af249af2845d0f0d37501aa688ffe5563</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1989</creationdate><topic>Amino acids</topic><topic>Analytical, structural and metabolic biochemistry</topic><topic>Arginine</topic><topic>Bacterial Proteins - ultrastructure</topic><topic>Binding Sites</topic><topic>Biological and medical sciences</topic><topic>Cell culture techniques</topic><topic>Curves</topic><topic>Deoxyribonuclease EcoRI - genetics</topic><topic>Deoxyribonuclease EcoRI - metabolism</topic><topic>DNA</topic><topic>DNA cleavage</topic><topic>DNA Mutational Analysis</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>DNA-Binding Proteins - ultrastructure</topic><topic>Enzymes</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - growth & development</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Hydrogen Bonding</topic><topic>Hydrogen bonds</topic><topic>Hydrolases</topic><topic>Models, Molecular</topic><topic>Phenotypes</topic><topic>Plasmids</topic><topic>Protein Conformation</topic><topic>Structure-Activity Relationship</topic><topic>Substrate Specificity</topic><topic>Viability</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Needels, M. C.</creatorcontrib><creatorcontrib>Fried, S. R.</creatorcontrib><creatorcontrib>Love, R.</creatorcontrib><creatorcontrib>Rosenberg, J. M.</creatorcontrib><creatorcontrib>Boyer, H. W.</creatorcontrib><creatorcontrib>Greene, P. 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J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Determinants of EcoRI Endonuclease Sequence Discrimination</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1989-05-01</date><risdate>1989</risdate><volume>86</volume><issue>10</issue><spage>3579</spage><epage>3583</epage><pages>3579-3583</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><coden>PNASA6</coden><abstract>The arginine at position 200 of EcoRI endonuclease is thought to make two hydrogen bonds to the guanine of the sequence GAATTC and thus be an important determinant of sequence discrimination. Arg-200 was replaced by each of the other 19 naturally occurring amino acids, and the mutant endonucleases were assessed for activities in vivo and in vitro. The mutant endonuclease with lysine at position 200 exhibits the most in vivo activity of all the position 200 mutants, although the in vitro activity is less than 1/100th of wild-type activity. Five other mutants show more drastically reduced levels of in vivo activity (Cys, Pro, Val, Ser, and Trp). The Cys, Val, and Ser mutant enzymes appear to have in vivo activity which is specific for the wild-type canonical site despite the loss of hydrogen bonding potential at position 200. The Pro and Trp mutants retain in vivo activity which is independent of the presence of the EcoRI methylase. In crude cell lysates, only the Cys mutant shows a very low level of in vitro activity. None of the mutant enzymes show a preference for alternative sites in assays in vitro. The implications of these results are discussed.</abstract><cop>Washington, DC</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>2657723</pmid><doi>10.1073/pnas.86.10.3579</doi><tpages>5</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; JSTOR Archive Collection A-Z Listing; PubMed Central; Alma/SFX Local Collection; Free Full-Text Journals in Chemistry |
| subjects | Amino acids Analytical, structural and metabolic biochemistry Arginine Bacterial Proteins - ultrastructure Binding Sites Biological and medical sciences Cell culture techniques Curves Deoxyribonuclease EcoRI - genetics Deoxyribonuclease EcoRI - metabolism DNA DNA cleavage DNA Mutational Analysis DNA-Binding Proteins - metabolism DNA-Binding Proteins - ultrastructure Enzymes Enzymes and enzyme inhibitors Escherichia coli - enzymology Escherichia coli - growth & development Fundamental and applied biological sciences. Psychology Hydrogen Bonding Hydrogen bonds Hydrolases Models, Molecular Phenotypes Plasmids Protein Conformation Structure-Activity Relationship Substrate Specificity Viability |
| title | Determinants of EcoRI Endonuclease Sequence Discrimination |
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