Structural Mechanism for Coordination of Proofreading and Polymerase Activities in Archaeal DNA Polymerases
A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3′-5′exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were a...
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| Veröffentlicht in: | Journal of molecular biology 2005-08, Vol.351 (2), p.291-298 |
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| creator | Kuroita, Toshihiro Matsumura, Hiroyoshi Yokota, Naohiko Kitabayashi, Masao Hashimoto, Hiroshi Inoue, Tsuyoshi Imanaka, Tadayuki Kai, Yasushi |
| description | A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3′-5′exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from
Thermococcus kodakaraensis KOD1 (previously
Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a “unique loop” in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3′-5′exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75
Å crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3′-5′exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases. |
| doi_str_mv | 10.1016/j.jmb.2005.06.015 |
| format | Article |
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Thermococcus kodakaraensis KOD1 (previously
Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a “unique loop” in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3′-5′exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75
Å crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3′-5′exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.</description><identifier>ISSN: 0022-2836</identifier><identifier>EISSN: 1089-8638</identifier><identifier>DOI: 10.1016/j.jmb.2005.06.015</identifier><identifier>PMID: 16019029</identifier><language>eng</language><publisher>England: Elsevier Ltd</publisher><subject>Amino Acid Sequence ; Animals ; Archaeal Proteins - chemistry ; Archaeal Proteins - physiology ; archaeral DNA polymerases ; Binding Sites ; Cattle ; Crystallography, X-Ray ; DNA - chemistry ; DNA-Directed DNA Polymerase - chemistry ; DNA-Directed DNA Polymerase - physiology ; domain interface ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; PCR performance ; Polymerase Chain Reaction ; proofreading activity ; Protein Conformation ; Protein Structure, Tertiary ; Static Electricity ; structural change ; Thermococcus - enzymology</subject><ispartof>Journal of molecular biology, 2005-08, Vol.351 (2), p.291-298</ispartof><rights>2005 Elsevier Ltd</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c492t-36387ee5521fc4fb06f5e7e3cbc09b0b8b36a39e20bbb02cc053be0edbf5e7d83</citedby><cites>FETCH-LOGICAL-c492t-36387ee5521fc4fb06f5e7e3cbc09b0b8b36a39e20bbb02cc053be0edbf5e7d83</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jmb.2005.06.015$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16019029$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kuroita, Toshihiro</creatorcontrib><creatorcontrib>Matsumura, Hiroyoshi</creatorcontrib><creatorcontrib>Yokota, Naohiko</creatorcontrib><creatorcontrib>Kitabayashi, Masao</creatorcontrib><creatorcontrib>Hashimoto, Hiroshi</creatorcontrib><creatorcontrib>Inoue, Tsuyoshi</creatorcontrib><creatorcontrib>Imanaka, Tadayuki</creatorcontrib><creatorcontrib>Kai, Yasushi</creatorcontrib><title>Structural Mechanism for Coordination of Proofreading and Polymerase Activities in Archaeal DNA Polymerases</title><title>Journal of molecular biology</title><addtitle>J Mol Biol</addtitle><description>A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3′-5′exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from
Thermococcus kodakaraensis KOD1 (previously
Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a “unique loop” in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3′-5′exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75
Å crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3′-5′exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Archaeal Proteins - chemistry</subject><subject>Archaeal Proteins - physiology</subject><subject>archaeral DNA polymerases</subject><subject>Binding Sites</subject><subject>Cattle</subject><subject>Crystallography, X-Ray</subject><subject>DNA - chemistry</subject><subject>DNA-Directed DNA Polymerase - chemistry</subject><subject>DNA-Directed DNA Polymerase - physiology</subject><subject>domain interface</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Mutation</subject><subject>PCR performance</subject><subject>Polymerase Chain Reaction</subject><subject>proofreading activity</subject><subject>Protein Conformation</subject><subject>Protein Structure, Tertiary</subject><subject>Static Electricity</subject><subject>structural change</subject><subject>Thermococcus - enzymology</subject><issn>0022-2836</issn><issn>1089-8638</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2005</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kMlOwzAQQC0EomX5AC7IJ24JY7tJE3GqyiqVRQLOlu1MwCWJwU4q8fe4aiU4cbI0evPkeYScMEgZsPx8mS5bnXKALIU8BZbtkDGDokyKXBS7ZAzAecILkY_IQQhLiKCYFPtkxHJgJfByTD6eez-YfvCqofdo3lVnQ0tr5-ncOV_ZTvXWddTV9Mk7V3tUcfZGVVfRJ9d8t-hVQDozvV3Z3mKgtqMzHz0YhZcPsz9UOCJ7tWoCHm_fQ_J6ffUyv00Wjzd389kiMZOS94mIn58iZhlntZnUGvI6wykKow2UGnShRa5EiRy01sCNiVdpBKz0mqsKcUjONt5P774GDL1sbTDYNKpDNwTJpiLjUGQRZBvQeBeCx1p-etsq_y0ZyHVhuZSxsFwXlpDLWDjunG7lg26x-t3YJo3AxQbAeOLKopfBWOwMVtaj6WXl7D_6H1sHjf4</recordid><startdate>20050812</startdate><enddate>20050812</enddate><creator>Kuroita, Toshihiro</creator><creator>Matsumura, Hiroyoshi</creator><creator>Yokota, Naohiko</creator><creator>Kitabayashi, Masao</creator><creator>Hashimoto, Hiroshi</creator><creator>Inoue, Tsuyoshi</creator><creator>Imanaka, Tadayuki</creator><creator>Kai, Yasushi</creator><general>Elsevier Ltd</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>7QL</scope><scope>7TM</scope><scope>C1K</scope></search><sort><creationdate>20050812</creationdate><title>Structural Mechanism for Coordination of Proofreading and Polymerase Activities in Archaeal DNA Polymerases</title><author>Kuroita, Toshihiro ; 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The 3′-5′exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from
Thermococcus kodakaraensis KOD1 (previously
Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a “unique loop” in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3′-5′exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75
Å crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3′-5′exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.</abstract><cop>England</cop><pub>Elsevier Ltd</pub><pmid>16019029</pmid><doi>10.1016/j.jmb.2005.06.015</doi><tpages>8</tpages></addata></record> |
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| subjects | Amino Acid Sequence Animals Archaeal Proteins - chemistry Archaeal Proteins - physiology archaeral DNA polymerases Binding Sites Cattle Crystallography, X-Ray DNA - chemistry DNA-Directed DNA Polymerase - chemistry DNA-Directed DNA Polymerase - physiology domain interface Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed Mutation PCR performance Polymerase Chain Reaction proofreading activity Protein Conformation Protein Structure, Tertiary Static Electricity structural change Thermococcus - enzymology |
| title | Structural Mechanism for Coordination of Proofreading and Polymerase Activities in Archaeal DNA Polymerases |
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