Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.

Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.

We report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3'-5' exonuclease domain. HoLaMa was overexpressed in Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR...

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Main Authors: Michael Kovermann, Alessandra Stefan, Anna Castaldo, Sara Caramia, Alejandro Hochkoeppler
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2019-01-01
Series:PLoS ONE
Online Access:https://doi.org/10.1371/journal.pone.0215411
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spelling doaj-cf506e5f50cd4f31b3cfc778871564e02021-06-19T05:10:34ZengPublic Library of Science (PLoS)PLoS ONE1932-62032019-01-01144e021541110.1371/journal.pone.0215411Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.Michael KovermannAlessandra StefanAnna CastaldoSara CaramiaAlejandro HochkoepplerWe report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3'-5' exonuclease domain. HoLaMa was overexpressed in Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20°C. In addition, urea induced a cooperative folding to unfolding transition of HoLaMa, possessing an overall thermodynamic stability and a transition midpoint featuring ΔG and CM equal to (15.7 ± 1.9) kJ/mol and (3.5 ± 0.6) M, respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme, we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly, HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs.https://doi.org/10.1371/journal.pone.0215411
collection DOAJ
language English
format Article
sources DOAJ
author Michael Kovermann
Alessandra Stefan
Anna Castaldo
Sara Caramia
Alejandro Hochkoeppler
spellingShingle Michael Kovermann
Alessandra Stefan
Anna Castaldo
Sara Caramia
Alejandro Hochkoeppler
Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.
PLoS ONE
author_facet Michael Kovermann
Alessandra Stefan
Anna Castaldo
Sara Caramia
Alejandro Hochkoeppler
author_sort Michael Kovermann
title Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.
title_short Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.
title_full Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.
title_fullStr Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.
title_full_unstemmed Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain.
title_sort structural and catalytic insights into holama, a derivative of klenow dna polymerase lacking the proofreading domain.
publisher Public Library of Science (PLoS)
series PLoS ONE
issn 1932-6203
publishDate 2019-01-01
description We report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3'-5' exonuclease domain. HoLaMa was overexpressed in Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20°C. In addition, urea induced a cooperative folding to unfolding transition of HoLaMa, possessing an overall thermodynamic stability and a transition midpoint featuring ΔG and CM equal to (15.7 ± 1.9) kJ/mol and (3.5 ± 0.6) M, respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme, we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly, HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs.
url https://doi.org/10.1371/journal.pone.0215411
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