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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

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KOVERMANN, Michael, Alessandra STEFAN, Anna CASTALDO, Sara CARAMIA, Alejandro HOCHKOEPPLER, 2019. Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain. In: PLoS one. 14(4), e0215411. eISSN 1932-6203. Available under: doi: 10.1371/journal.pone.0215411

@article{Kovermann2019Struc-46067, title={Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain}, year={2019}, doi={10.1371/journal.pone.0215411}, number={4}, volume={14}, journal={PLoS one}, author={Kovermann, Michael and Stefan, Alessandra and Castaldo, Anna and Caramia, Sara and Hochkoeppler, Alejandro}, note={Article Number: e0215411} }

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 C<sub>M</sub> 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. Kovermann, Michael Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain 2019 2019-06-21T12:28:24Z Stefan, Alessandra Castaldo, Anna Hochkoeppler, Alejandro eng Caramia, Sara 2019-06-21T12:28:24Z terms-of-use Castaldo, Anna Stefan, Alessandra Kovermann, Michael Caramia, Sara Hochkoeppler, Alejandro

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