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Mutational analysis of human DNase I at the DNA binding interface: Implications for DNA recognition, catalysis, and metal ion dependence
Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site‐directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the partic...
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Published in: | Protein science 1998-03, Vol.7 (3), p.628-636 |
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description | Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site‐directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion‐coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gin 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I‐like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I‐like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+‐dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions. |
doi_str_mv | 10.1002/pro.5560070312 |
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Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion‐coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gin 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I‐like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I‐like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+‐dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.</description><identifier>ISSN: 0961-8368</identifier><identifier>EISSN: 1469-896X</identifier><identifier>DOI: 10.1002/pro.5560070312</identifier><identifier>PMID: 9541395</identifier><language>eng</language><publisher>Bristol: Cold Spring Harbor Laboratory Press</publisher><subject>Animals ; Binding Sites ; Catalysis ; Cations, Divalent ; Cattle ; Deoxyribonuclease I - chemistry ; Deoxyribonuclease I - metabolism ; DNA Mutational Analysis ; DNA-Binding Proteins - chemistry ; DNA-Binding Proteins - metabolism ; DNase I ; Histidine - chemistry ; Humans ; Hydrogen Bonding ; Metals ; Models, Molecular ; Plasmids ; protein‐DNA interactions ; Recombinant Proteins ; site‐directed mutagenesis ; Species Specificity ; Structure-Activity Relationship ; structure‐function analysis</subject><ispartof>Protein science, 1998-03, Vol.7 (3), p.628-636</ispartof><rights>Copyright © 1998 The Protein Society</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c5012-5aa9089fa5a191b028e86e3dcb1844cb5eddc5e9befe7a42cc81b7ca8ac2bb2f3</citedby><cites>FETCH-LOGICAL-c5012-5aa9089fa5a191b028e86e3dcb1844cb5eddc5e9befe7a42cc81b7ca8ac2bb2f3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143959/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2143959/$$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/9541395$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Pan, Clark Q.</creatorcontrib><creatorcontrib>Uumer, Jana S.</creatorcontrib><creatorcontrib>Herzka, Andrea</creatorcontrib><creatorcontrib>Lazarus, Robert A.</creatorcontrib><title>Mutational analysis of human DNase I at the DNA binding interface: Implications for DNA recognition, catalysis, and metal ion dependence</title><title>Protein science</title><addtitle>Protein Sci</addtitle><description>Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site‐directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion‐coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gin 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I‐like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I‐like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+‐dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.</description><subject>Animals</subject><subject>Binding Sites</subject><subject>Catalysis</subject><subject>Cations, Divalent</subject><subject>Cattle</subject><subject>Deoxyribonuclease I - chemistry</subject><subject>Deoxyribonuclease I - metabolism</subject><subject>DNA Mutational Analysis</subject><subject>DNA-Binding Proteins - chemistry</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>DNase I</subject><subject>Histidine - chemistry</subject><subject>Humans</subject><subject>Hydrogen Bonding</subject><subject>Metals</subject><subject>Models, Molecular</subject><subject>Plasmids</subject><subject>protein‐DNA interactions</subject><subject>Recombinant Proteins</subject><subject>site‐directed mutagenesis</subject><subject>Species Specificity</subject><subject>Structure-Activity Relationship</subject><subject>structure‐function analysis</subject><issn>0961-8368</issn><issn>1469-896X</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><recordid>eNqFkcFO3DAQhi1URLeUKzckPwBZbCfO2j1UQrSFlaBUiEq9RWNnsusqsSM722rfoI9dwyKgp15m9Puf-UbyT8gxZ3POmDgbY5hLWTO2YCUXe2TGq1oXStc_3pAZ0zUvVFmrt-RdSj8ZYxUX5QE50LLipZYz8udmM8HkgoeeQi7b5BINHV1vBvD001dISJcUJjqtMctzapxvnV9R5yeMHVj8QJfD2Dv7SEm0C_FxLqINK-8eHk9pNnfo03ykpQNmSbNDWxzRt-gtvif7HfQJj576Ifn-5fP9xVVxfXu5vDi_LqxkXBQSQDOlO5DANTdMKFQ1lq01XFWVNRLb1krUBjtcQCWsVdwsLCiwwhjRlYfk4447bsyArUU_ReibMboB4rYJ4Jp_He_WzSr8agSv8o_pDJjvADaGlCJ2z7ucNQ-RZB2al0jywsnri8_jTxlkX-_8367H7X9ozbe721fsvyJvnF8</recordid><startdate>199803</startdate><enddate>199803</enddate><creator>Pan, Clark Q.</creator><creator>Uumer, Jana S.</creator><creator>Herzka, Andrea</creator><creator>Lazarus, Robert A.</creator><general>Cold Spring Harbor Laboratory Press</general><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>5PM</scope></search><sort><creationdate>199803</creationdate><title>Mutational analysis of human DNase I at the DNA binding interface: Implications for DNA recognition, catalysis, and metal ion dependence</title><author>Pan, Clark Q. ; Uumer, Jana S. ; Herzka, Andrea ; Lazarus, Robert A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c5012-5aa9089fa5a191b028e86e3dcb1844cb5eddc5e9befe7a42cc81b7ca8ac2bb2f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Animals</topic><topic>Binding Sites</topic><topic>Catalysis</topic><topic>Cations, Divalent</topic><topic>Cattle</topic><topic>Deoxyribonuclease I - chemistry</topic><topic>Deoxyribonuclease I - metabolism</topic><topic>DNA Mutational Analysis</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>DNase I</topic><topic>Histidine - chemistry</topic><topic>Humans</topic><topic>Hydrogen Bonding</topic><topic>Metals</topic><topic>Models, Molecular</topic><topic>Plasmids</topic><topic>protein‐DNA interactions</topic><topic>Recombinant Proteins</topic><topic>site‐directed mutagenesis</topic><topic>Species Specificity</topic><topic>Structure-Activity Relationship</topic><topic>structure‐function analysis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pan, Clark Q.</creatorcontrib><creatorcontrib>Uumer, Jana S.</creatorcontrib><creatorcontrib>Herzka, Andrea</creatorcontrib><creatorcontrib>Lazarus, Robert A.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Protein science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Pan, Clark Q.</au><au>Uumer, Jana S.</au><au>Herzka, Andrea</au><au>Lazarus, Robert A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Mutational analysis of human DNase I at the DNA binding interface: Implications for DNA recognition, catalysis, and metal ion dependence</atitle><jtitle>Protein science</jtitle><addtitle>Protein Sci</addtitle><date>1998-03</date><risdate>1998</risdate><volume>7</volume><issue>3</issue><spage>628</spage><epage>636</epage><pages>628-636</pages><issn>0961-8368</issn><eissn>1469-896X</eissn><abstract>Human deoxyribonuclease I (DNase I), an enzyme used to treat cystic fibrosis patients, has been systematically analyzed by site‐directed mutagenesis of residues at the DNA binding interface. Crystal structures of bovine DNase I complexed with two different oligonucleotides have implicated the participation of over 20 amino acids in catalysis or DNA recognition. These residues have been classified into four groups based on the characterization of over 80 human DNase I variants. Mutations at any of the four catalytic amino acids His 134, His 252, Glu 78, and Asp 212 drastically reduced the hydrolytic activity of DNase I. Replacing the three putative divalent metal ion‐coordinating residues Glu 39, Asp 168, or Asp 251 led to inactive variants. Amino acids Gin 9, Arg 41, Tyr 76, Arg 111, Asn 170, Tyr 175, and Tyr 211 were also critical for activity, presumably because of their close proximity to the active site, while more peripheral DNA interactions stemming from 13 other positions were of minimal significance. The relative importance of these 27 positions is consistent with evolutionary relationships among DNase I across different species, DNase I‐like proteins, and bacterial sphingomyelinases, suggesting a fingerprint for a family of DNase I‐like proteins. Furthermore, we found no evidence for a second active site that had been previously implicated in Mn2+‐dependent DNA degradation. Finally, we correlated our mutational analysis of human DNase I to that of bovine DNase I with respect to their specific activity and dependence on divalent metal ions.</abstract><cop>Bristol</cop><pub>Cold Spring Harbor Laboratory Press</pub><pmid>9541395</pmid><doi>10.1002/pro.5560070312</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Animals Binding Sites Catalysis Cations, Divalent Cattle Deoxyribonuclease I - chemistry Deoxyribonuclease I - metabolism DNA Mutational Analysis DNA-Binding Proteins - chemistry DNA-Binding Proteins - metabolism DNase I Histidine - chemistry Humans Hydrogen Bonding Metals Models, Molecular Plasmids protein‐DNA interactions Recombinant Proteins site‐directed mutagenesis Species Specificity Structure-Activity Relationship structure‐function analysis |
title | Mutational analysis of human DNase I at the DNA binding interface: Implications for DNA recognition, catalysis, and metal ion dependence |
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