Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis

The side chain of Val75 stabilizes the fingers subdomain of the human immunodeficiency virus type 1 reverse transcriptase (RT), while its peptide backbone interacts with the single-stranded DNA template (at nucleotide + 1) and with the peptide backbone of Gln151. Specific DNA polymerase activities o...

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Detalles Bibliográficos
Autores: Matamoros Grande, Tania, Kim, Baek, Menéndez-Arias, Luis
Tipo de recurso: artículo
Fecha de publicación:2007
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/4925
Acceso en línea:http://hdl.handle.net/10261/4925
Access Level:acceso abierto
Palabra clave:HIV
DNA polymerase
Fidelity
Reverse transcriptase
Drug resistance
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spelling Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA SynthesisMatamoros Grande, TaniaKim, BaekMenéndez-Arias, LuisHIVDNA polymeraseFidelityReverse transcriptaseDrug resistanceThe side chain of Val75 stabilizes the fingers subdomain of the human immunodeficiency virus type 1 reverse transcriptase (RT), while its peptide backbone interacts with the single-stranded DNA template (at nucleotide + 1) and with the peptide backbone of Gln151. Specific DNA polymerase activities of mutant RTs bearing amino acid substitutions at position 75 (i.e., V75A, V75F, V75I, V75L, V75M, V75S and V75T) were relatively high. Primer extension experiments carried out in the absence of one deoxyribonucleoside-triphosphate suggested that mutations did not affect the accuracy of the RT, except for V75A, V75F, V75I, and to a lesser extent V75T. The fidelity of RTs bearing mutations V75F and V75I increased 1.8- and 3-fold, respectively, as measured by the M13 lacZ forward mutation assay, while V75A showed 1.4-fold decreased accuracy. Steady- and pre-steady-state kinetics demonstrated that the increased fidelity of V75I and V75F was related to their decreased ability to extend mismatched template–primers, while misincorporation efficiencies were not significantly affected by mutations. The increased mispair extension fidelity of mutant V75I RT could be attributed to the nucleotide affinity loss, observed in reactions with mismatched template–primers. Altogether, these data suggest that Val75 interactions with the 5′ template overhang are important determinants of fidelityWork in Madrid was supported in part by grants of the Spanish Ministry of Education and Science (BIO2003/01175), Fondo de Investigación Sanitaria (through “Red Temática de Investigación Cooperativa en SIDA” RD06/0006), and an institutional grant of Fundación Ramón Areces. T.M. is a recipient of a predoctoral fellowship of the Spanish Ministry of Education and SciencePeer reviewedElsevierMinisterio de Educación y Ciencia (España)Instituto de Salud Carlos IIIFundación Ramón Areces200820082007info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501452440 bytesapplication/pdfhttp://hdl.handle.net/10261/4925reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttp://dx.doi.org/10.1016/j.jmb.2007.11.021info:eu-repo/semantics/openAccessoai:digital.csic.es:10261/49252026-05-22T06:33:51Z
dc.title.none.fl_str_mv Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis
title Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis
spellingShingle Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis
Matamoros Grande, Tania
HIV
DNA polymerase
Fidelity
Reverse transcriptase
Drug resistance
title_short Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis
title_full Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis
title_fullStr Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis
title_full_unstemmed Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis
title_sort Mechanistic Insights into the Role of Val75 of HIV-1 Reverse Transcriptase in Misinsertion and Mispair Extension Fidelity of DNA Synthesis
dc.creator.none.fl_str_mv Matamoros Grande, Tania
Kim, Baek
Menéndez-Arias, Luis
author Matamoros Grande, Tania
author_facet Matamoros Grande, Tania
Kim, Baek
Menéndez-Arias, Luis
author_role author
author2 Kim, Baek
Menéndez-Arias, Luis
author2_role author
author
dc.contributor.none.fl_str_mv Ministerio de Educación y Ciencia (España)
Instituto de Salud Carlos III
Fundación Ramón Areces
dc.subject.none.fl_str_mv HIV
DNA polymerase
Fidelity
Reverse transcriptase
Drug resistance
topic HIV
DNA polymerase
Fidelity
Reverse transcriptase
Drug resistance
description The side chain of Val75 stabilizes the fingers subdomain of the human immunodeficiency virus type 1 reverse transcriptase (RT), while its peptide backbone interacts with the single-stranded DNA template (at nucleotide + 1) and with the peptide backbone of Gln151. Specific DNA polymerase activities of mutant RTs bearing amino acid substitutions at position 75 (i.e., V75A, V75F, V75I, V75L, V75M, V75S and V75T) were relatively high. Primer extension experiments carried out in the absence of one deoxyribonucleoside-triphosphate suggested that mutations did not affect the accuracy of the RT, except for V75A, V75F, V75I, and to a lesser extent V75T. The fidelity of RTs bearing mutations V75F and V75I increased 1.8- and 3-fold, respectively, as measured by the M13 lacZ forward mutation assay, while V75A showed 1.4-fold decreased accuracy. Steady- and pre-steady-state kinetics demonstrated that the increased fidelity of V75I and V75F was related to their decreased ability to extend mismatched template–primers, while misincorporation efficiencies were not significantly affected by mutations. The increased mispair extension fidelity of mutant V75I RT could be attributed to the nucleotide affinity loss, observed in reactions with mismatched template–primers. Altogether, these data suggest that Val75 interactions with the 5′ template overhang are important determinants of fidelity
publishDate 2007
dc.date.none.fl_str_mv 2007
2008
2008
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/4925
url http://hdl.handle.net/10261/4925
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv http://dx.doi.org/10.1016/j.jmb.2007.11.021
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 452440 bytes
application/pdf
dc.publisher.none.fl_str_mv Elsevier
publisher.none.fl_str_mv Elsevier
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
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