Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

Abstract Non-natural nucleotide substrates are widely used in the enzymatic synthesis of modified DNA. The terminal activity of polymerases in the presence of modified nucleotides is an important, but poorly characterized, aspect of enzymatic DNA synthesis. Here, we studied different types of polyme...

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Autores principales:	Igor P. Smirnov, Natalia A. Kolganova, Vadim A. Vasiliskov, Alexander V. Chudinov, Edward N. Timofeev
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5c05e5230b0b47f886d7827c15df7e11
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spelling	oai:doaj.org-article:5c05e5230b0b47f886d7827c15df7e112021-12-02T16:08:11ZMass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases10.1038/s41598-017-06136-92045-2322https://doaj.org/article/5c05e5230b0b47f886d7827c15df7e112017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-06136-9https://doaj.org/toc/2045-2322Abstract Non-natural nucleotide substrates are widely used in the enzymatic synthesis of modified DNA. The terminal activity of polymerases in the presence of modified nucleotides is an important, but poorly characterized, aspect of enzymatic DNA synthesis. Here, we studied different types of polymerase activity at sequence ends using extendable and non-extendable synthetic models in the presence of the Cy5-dUTP analog Y. In primer extension reactions with selected exonuclease-deficient polymerases, nucleotide Y appeared to be a preferential substrate for non-templated 3′-tailing, as determined by MALDI mass-spectrometry and gel-electrophoresis. This result was further confirmed by the 3′-tailing of a non-extendable hairpin oligonucleotide model. Additionally, DNA polymerases induce an exchange of the 3′ terminal thymidine for a non-natural nucleotide via pyrophosphorolysis in the presence of inorganic pyrophosphate. In primer extension reactions, the proofreading polymerases Vent, Pfu, and Phusion did not support the synthesis of Y-modified primer strand. Nevertheless, Pfu and Phusion polymerases were shown to initiate terminal nucleotide exchange at the template. Unlike non-proofreading polymerases, these two enzymes recruit 3′–5′ exonuclease functions to cleave the 3′ terminal thymidine in the absence of pyrophosphate.Igor P. SmirnovNatalia A. KolganovaVadim A. VasiliskovAlexander V. ChudinovEdward N. TimofeevNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Igor P. Smirnov Natalia A. Kolganova Vadim A. Vasiliskov Alexander V. Chudinov Edward N. Timofeev Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases
description	Abstract Non-natural nucleotide substrates are widely used in the enzymatic synthesis of modified DNA. The terminal activity of polymerases in the presence of modified nucleotides is an important, but poorly characterized, aspect of enzymatic DNA synthesis. Here, we studied different types of polymerase activity at sequence ends using extendable and non-extendable synthetic models in the presence of the Cy5-dUTP analog Y. In primer extension reactions with selected exonuclease-deficient polymerases, nucleotide Y appeared to be a preferential substrate for non-templated 3′-tailing, as determined by MALDI mass-spectrometry and gel-electrophoresis. This result was further confirmed by the 3′-tailing of a non-extendable hairpin oligonucleotide model. Additionally, DNA polymerases induce an exchange of the 3′ terminal thymidine for a non-natural nucleotide via pyrophosphorolysis in the presence of inorganic pyrophosphate. In primer extension reactions, the proofreading polymerases Vent, Pfu, and Phusion did not support the synthesis of Y-modified primer strand. Nevertheless, Pfu and Phusion polymerases were shown to initiate terminal nucleotide exchange at the template. Unlike non-proofreading polymerases, these two enzymes recruit 3′–5′ exonuclease functions to cleave the 3′ terminal thymidine in the absence of pyrophosphate.
format	article
author	Igor P. Smirnov Natalia A. Kolganova Vadim A. Vasiliskov Alexander V. Chudinov Edward N. Timofeev
author_facet	Igor P. Smirnov Natalia A. Kolganova Vadim A. Vasiliskov Alexander V. Chudinov Edward N. Timofeev
author_sort	Igor P. Smirnov
title	Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases
title_short	Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases
title_full	Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases
title_fullStr	Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases
title_full_unstemmed	Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases
title_sort	mass-spectrometry analysis of modifications at dna termini induced by dna polymerases
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/5c05e5230b0b47f886d7827c15df7e11
work_keys_str_mv	AT igorpsmirnov massspectrometryanalysisofmodificationsatdnaterminiinducedbydnapolymerases AT nataliaakolganova massspectrometryanalysisofmodificationsatdnaterminiinducedbydnapolymerases AT vadimavasiliskov massspectrometryanalysisofmodificationsatdnaterminiinducedbydnapolymerases AT alexandervchudinov massspectrometryanalysisofmodificationsatdnaterminiinducedbydnapolymerases AT edwardntimofeev massspectrometryanalysisofmodificationsatdnaterminiinducedbydnapolymerases
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Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases

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