Fidelity analysis of HIV‐1 reverse transcriptase mutants with an altered amino‐acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184
Substitution of particular residues postulated to have a role in active site architecture can alter the overall fidelity of DNA polymerization by HIV‐1. The effects of this kind of substitution were determined in a lacZ‐based assay using HIV‐1 reverse transcriptase with specifically mutated residues...
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Veröffentlicht in: | European journal of biochemistry 2000-05, Vol.267 (9), p.2658-2665 |
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description | Substitution of particular residues postulated to have a role in active site architecture can alter the overall fidelity of DNA polymerization by HIV‐1. The effects of this kind of substitution were determined in a lacZ‐based assay using HIV‐1 reverse transcriptase with specifically mutated residues. We found that the reported higher fidelity of nucleotide incorporation by the Met184→Val and Glu89→Gly mutant reverse transcriptases (RTs) was not reflected in a substantial increase in the overall fidelity for these RT mutants. For the 3TC‐resistant Met184→Val RT mutant an almost wild‐type level of overall mutation frequency was observed, while the foscarnet‐resistant RTs harbouring the Glu89→Gly mutation showed only a twofold decrease in mutation frequency. The Tyr183→Phe mutant RT displayed a slightly lower fidelity than wild‐type RT. Conversely, the ddI‐resistant RT mutant containing the Leu74→Val mutation showed a 3.5‐fold higher fidelity compared to the wild‐type enzyme. Finally, the Tyr115→Ala substitution rendered the enzyme substantially more error‐prone for DNA polymerization. These results correlate with three‐dimensional structural studies of the polymerase active site and confirm the postulated impact of the Leu74, Tyr183 and Tyr115 RT residues on the overall fidelity of DNA polymerization by HIV‐1 RT. |
doi_str_mv | 10.1046/j.1432-1327.2000.01272.x |
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The effects of this kind of substitution were determined in a lacZ‐based assay using HIV‐1 reverse transcriptase with specifically mutated residues. We found that the reported higher fidelity of nucleotide incorporation by the Met184→Val and Glu89→Gly mutant reverse transcriptases (RTs) was not reflected in a substantial increase in the overall fidelity for these RT mutants. For the 3TC‐resistant Met184→Val RT mutant an almost wild‐type level of overall mutation frequency was observed, while the foscarnet‐resistant RTs harbouring the Glu89→Gly mutation showed only a twofold decrease in mutation frequency. The Tyr183→Phe mutant RT displayed a slightly lower fidelity than wild‐type RT. Conversely, the ddI‐resistant RT mutant containing the Leu74→Val mutation showed a 3.5‐fold higher fidelity compared to the wild‐type enzyme. Finally, the Tyr115→Ala substitution rendered the enzyme substantially more error‐prone for DNA polymerization. These results correlate with three‐dimensional structural studies of the polymerase active site and confirm the postulated impact of the Leu74, Tyr183 and Tyr115 RT residues on the overall fidelity of DNA polymerization by HIV‐1 RT.</description><identifier>ISSN: 0014-2956</identifier><identifier>EISSN: 1432-1033</identifier><identifier>DOI: 10.1046/j.1432-1327.2000.01272.x</identifier><identifier>PMID: 10785387</identifier><language>eng</language><publisher>Oxford, UK: Blackwell Science Ltd</publisher><subject>Amino Acid Substitution ; Amino Acids - chemistry ; Amino Acids - genetics ; Base Sequence ; Biopolymers ; fidelity ; HIV Reverse Transcriptase - chemistry ; HIV Reverse Transcriptase - genetics ; HIV Reverse Transcriptase - metabolism ; HIV‐1 RT ; Human immunodeficiency virus 1 ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; mutant reverse transcriptase ; polymerase fidelity ; polymerization ; Sequence Homology, Nucleic Acid ; site‐directed mutagenesis</subject><ispartof>European journal of biochemistry, 2000-05, Vol.267 (9), p.2658-2665</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4472-78ecf38db4950a97fcc9ed5370892b3ed34a898bc29c2bd0e1fd6d1eebb396cd3</citedby><cites>FETCH-LOGICAL-c4472-78ecf38db4950a97fcc9ed5370892b3ed34a898bc29c2bd0e1fd6d1eebb396cd3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1046%2Fj.1432-1327.2000.01272.x$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1046%2Fj.1432-1327.2000.01272.x$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,776,780,1411,27903,27904,45553,45554</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10785387$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Jonckheere, Heidi</creatorcontrib><creatorcontrib>De Clercq, Erik</creatorcontrib><creatorcontrib>Anné, Jozef</creatorcontrib><title>Fidelity analysis of HIV‐1 reverse transcriptase mutants with an altered amino‐acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184</title><title>European journal of biochemistry</title><addtitle>Eur J Biochem</addtitle><description>Substitution of particular residues postulated to have a role in active site architecture can alter the overall fidelity of DNA polymerization by HIV‐1. The effects of this kind of substitution were determined in a lacZ‐based assay using HIV‐1 reverse transcriptase with specifically mutated residues. We found that the reported higher fidelity of nucleotide incorporation by the Met184→Val and Glu89→Gly mutant reverse transcriptases (RTs) was not reflected in a substantial increase in the overall fidelity for these RT mutants. For the 3TC‐resistant Met184→Val RT mutant an almost wild‐type level of overall mutation frequency was observed, while the foscarnet‐resistant RTs harbouring the Glu89→Gly mutation showed only a twofold decrease in mutation frequency. The Tyr183→Phe mutant RT displayed a slightly lower fidelity than wild‐type RT. Conversely, the ddI‐resistant RT mutant containing the Leu74→Val mutation showed a 3.5‐fold higher fidelity compared to the wild‐type enzyme. Finally, the Tyr115→Ala substitution rendered the enzyme substantially more error‐prone for DNA polymerization. These results correlate with three‐dimensional structural studies of the polymerase active site and confirm the postulated impact of the Leu74, Tyr183 and Tyr115 RT residues on the overall fidelity of DNA polymerization by HIV‐1 RT.</description><subject>Amino Acid Substitution</subject><subject>Amino Acids - chemistry</subject><subject>Amino Acids - genetics</subject><subject>Base Sequence</subject><subject>Biopolymers</subject><subject>fidelity</subject><subject>HIV Reverse Transcriptase - chemistry</subject><subject>HIV Reverse Transcriptase - genetics</subject><subject>HIV Reverse Transcriptase - metabolism</subject><subject>HIV‐1 RT</subject><subject>Human immunodeficiency virus 1</subject><subject>Models, Molecular</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>mutant reverse transcriptase</subject><subject>polymerase fidelity</subject><subject>polymerization</subject><subject>Sequence Homology, Nucleic Acid</subject><subject>site‐directed mutagenesis</subject><issn>0014-2956</issn><issn>1432-1033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2000</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkc1u1DAYRS0EokPhFZBXrDrBP0lsb5Cg6rSVBrGgsLUc-4vwKD-D7bTNjkdgwRPyJDhNF12y-mz53GvLByFMSUFJWb8_FLTkbEs5EwUjhBSEMsGK-2dosx4Qzp-jDSG03DJV1SfoVYyHDNaqFi_RCSVCVlyKDfqz8w46n2ZsBtPN0Uc8tvjq-vvfX78pDnALIQJOwQzRBn9MJu_6KZkhRXzn048cw6ZLEMBh0_thzDljvcMRfk4wWMAm5Zro3QQR72ES5Rm-7CapzvDNHCit1il5bnL4MyQqy9foRWu6CG8e5yn6tru4Ob_a7r9cXp9_3G9tWQq2FRJsy6VrSlURo0RrrQJXcUGkYg0Hx0sjlWwsU5Y1jgBtXe0oQNNwVVvHT9G7tfcYxvzamHTvo4WuMwOMU9RU1LyiqsqgXEEbxhgDtPoYfG_CrCnRixF90MvH68WIXozoByP6PkffPt4xNT24J8FVQQY-rMCd72D-72K9u_j0dVnyf5POnCY</recordid><startdate>200005</startdate><enddate>200005</enddate><creator>Jonckheere, Heidi</creator><creator>De Clercq, Erik</creator><creator>Anné, Jozef</creator><general>Blackwell Science 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>7U9</scope><scope>H94</scope></search><sort><creationdate>200005</creationdate><title>Fidelity analysis of HIV‐1 reverse transcriptase mutants with an altered amino‐acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184</title><author>Jonckheere, Heidi ; De Clercq, Erik ; Anné, Jozef</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4472-78ecf38db4950a97fcc9ed5370892b3ed34a898bc29c2bd0e1fd6d1eebb396cd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2000</creationdate><topic>Amino Acid Substitution</topic><topic>Amino Acids - chemistry</topic><topic>Amino Acids - genetics</topic><topic>Base Sequence</topic><topic>Biopolymers</topic><topic>fidelity</topic><topic>HIV Reverse Transcriptase - chemistry</topic><topic>HIV Reverse Transcriptase - genetics</topic><topic>HIV Reverse Transcriptase - metabolism</topic><topic>HIV‐1 RT</topic><topic>Human immunodeficiency virus 1</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>mutant reverse transcriptase</topic><topic>polymerase fidelity</topic><topic>polymerization</topic><topic>Sequence Homology, Nucleic Acid</topic><topic>site‐directed mutagenesis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Jonckheere, Heidi</creatorcontrib><creatorcontrib>De Clercq, Erik</creatorcontrib><creatorcontrib>Anné, Jozef</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Virology and AIDS Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><jtitle>European journal of biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Jonckheere, Heidi</au><au>De Clercq, Erik</au><au>Anné, Jozef</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Fidelity analysis of HIV‐1 reverse transcriptase mutants with an altered amino‐acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184</atitle><jtitle>European journal of biochemistry</jtitle><addtitle>Eur J Biochem</addtitle><date>2000-05</date><risdate>2000</risdate><volume>267</volume><issue>9</issue><spage>2658</spage><epage>2665</epage><pages>2658-2665</pages><issn>0014-2956</issn><eissn>1432-1033</eissn><abstract>Substitution of particular residues postulated to have a role in active site architecture can alter the overall fidelity of DNA polymerization by HIV‐1. The effects of this kind of substitution were determined in a lacZ‐based assay using HIV‐1 reverse transcriptase with specifically mutated residues. We found that the reported higher fidelity of nucleotide incorporation by the Met184→Val and Glu89→Gly mutant reverse transcriptases (RTs) was not reflected in a substantial increase in the overall fidelity for these RT mutants. For the 3TC‐resistant Met184→Val RT mutant an almost wild‐type level of overall mutation frequency was observed, while the foscarnet‐resistant RTs harbouring the Glu89→Gly mutation showed only a twofold decrease in mutation frequency. The Tyr183→Phe mutant RT displayed a slightly lower fidelity than wild‐type RT. Conversely, the ddI‐resistant RT mutant containing the Leu74→Val mutation showed a 3.5‐fold higher fidelity compared to the wild‐type enzyme. Finally, the Tyr115→Ala substitution rendered the enzyme substantially more error‐prone for DNA polymerization. These results correlate with three‐dimensional structural studies of the polymerase active site and confirm the postulated impact of the Leu74, Tyr183 and Tyr115 RT residues on the overall fidelity of DNA polymerization by HIV‐1 RT.</abstract><cop>Oxford, UK</cop><pub>Blackwell Science Ltd</pub><pmid>10785387</pmid><doi>10.1046/j.1432-1327.2000.01272.x</doi><tpages>8</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Amino Acid Substitution Amino Acids - chemistry Amino Acids - genetics Base Sequence Biopolymers fidelity HIV Reverse Transcriptase - chemistry HIV Reverse Transcriptase - genetics HIV Reverse Transcriptase - metabolism HIV‐1 RT Human immunodeficiency virus 1 Models, Molecular Molecular Sequence Data Mutagenesis, Site-Directed mutant reverse transcriptase polymerase fidelity polymerization Sequence Homology, Nucleic Acid site‐directed mutagenesis |
title | Fidelity analysis of HIV‐1 reverse transcriptase mutants with an altered amino‐acid sequence at residues Leu74, Glu89, Tyr115, Tyr183 and Met184 |
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