From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase

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From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Figure 1. Catalytic activities of PabPolB mediated by Mg<sup>2+</sup> or Ca<sup>2+</sup>. (A) Primer-template used for primer extension and 3′-exonuclease primer degradation experiments, in panels B–D, respectively. It consists of a Cy5-labeled 17-mer primer annealed to a DNA template of 87-nt in length. (B) Extension of the 17/87 primer-template by PabPolB at the indicated Mg<sup>2+</sup> or Ca<sup>2+</sup> concentrations. The numbers under the gel lanes represent the total percentage of full-length products and extension. Reference oligodeoxynucleotides of 17 and 87 bases are indicated on the right. (C) Extension of the 17/87 primed-template (25 nM) by PabPolB (75 nM) at fixed Mg<sup>2+</sup> or Ca<sup>2+</sup> concentrations (5 mM) and 200 μM dNTPs in a time course experiment at 55°C. The numbers under the gel lanes represent the total percentage of full-length products. Reference oligodeoxynucleotides of 17 and 87 bases are indicated on the right. (D) Proofreading exonucleolysis of 17/87 primer-template at the indicated Mg<sup>2+</sup> or Ca<sup>2+</sup> concentrations. The numbers under the gel lanes represent the percentage of degraded primers. Reference oligodeoxynucleotides of 17 and 8 bases are indicated on the right. From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Nucleic Acids Res. 2017;45(21):12425-12440. doi:10.1093/nar/gkx927 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Figure 2. Modulation of DNA polymerization PabPolB with Mg<sup>2+</sup> or Ca<sup>2+</sup> on primed-M13mp18 DNA template. (A) Structure of the primer-template mimic, consisting of a Cy5-labeled 32-mer primer annealed to the circular M13mp18 DNA template of 7249-nt in length. (B) Extension of the primed-M13mp18 DNA template at the indicated Mg<sup>2+</sup> or Ca<sup>2+</sup> concentrations. The starting primer (32-nt) and full-length product (7249-nt) are shown arrowed on the left. Product length is indicated under the gel lanes. (C) Extension of the primed-M13mp18 DNA template for the times shown above the gels (min) at fixed Mg<sup>2+</sup> or Ca<sup>2+</sup> concentrations (5 mM). The starting primer (32-nt) and full-length product (7249-nt) are shown arrowed on the left. Product length is indicated under the gel lanes. From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Nucleic Acids Res. 2017;45(21):12425-12440. doi:10.1093/nar/gkx927 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Figure 3. Single nucleotide incorporation by PabPolB in the presence of Mg<sup>2+</sup> or Ca<sup>2+</sup>. (A) Primer-template used for these experiments consists of a Cy5-labeled 26-mer primer annealed to a DNA template of 34-nt in length. Incorporation using PabPolB exo+ (B) or PabPolB exo− (C) at 5 mM Mg<sup>2+</sup> or Ca<sup>2+</sup> concentrations. 0 = no dNTPs added; + = all four dNTPs added; A, T, C and G = only dATP or dTTP or dCTP or dGTP added, respectively. The extension (%) for selected lanes is shown under the gels. From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Nucleic Acids Res. 2017;45(21):12425-12440. doi:10.1093/nar/gkx927 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Figure 4. Ionic accessibility of polymerase and exonuclease active sites of PabPolB. (A) Primer-template used for 3′-exonuclease primer degradation and primer extension experiments, in panels B and C and D and E, respectively. It consists of a Cy5-labeled 17-mer primer annealed to a DNA template of 87-nt in length. Ion displacement experiment is carried out at fixed Mg<sup>2+</sup> and Ca<sup>2+</sup> concentrations and by increasing metal ion competitor concentrations, respectively, in panels B–D and C–E. PabPolB is pre-incubated at the indicated fixed metal concentrations. Chased experiment is initiated by co-addition of p/t (17/87) and increased ion competitor concentrations. Equimolar concentrations are shown framed. Reference oligodeoxynucleotides of 87, 17 and 8 bases are indicated on the right of each panel. From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Nucleic Acids Res. 2017;45(21):12425-12440. doi:10.1093/nar/gkx927 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Figure 5. Contribution of diverse metal ions to exonuclease and DNA polymerase activities by PabPolB. (A) Primer-template used for primer extension experiments and 3′-exonuclease primer degradation, in panels B and C, respectively. It consists of a Cy5-labeled 17-mer primer annealed to a DNA template of 87-nt in length. (B) Extension of the 17/87 primer-template by PabPolB at 5 mM metal ion concentrations. The numbers under the gel lanes represent the total percentage of extended products. Reference oligodeoxynucleotides of 17, 32, 58 and 87 bases are indicated by the arrows. Ionic radii (Å) are displayed above metal ions. (C) Proofreading exonucleolysis of 17/87 primer-template at 5 mM metal ion concentrations. The numbers under the gel lanes represent the percentage of degraded primers. Reference oligodeoxynucleotides of 17 and 8 bases are indicated by the arrows. From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Nucleic Acids Res. 2017;45(21):12425-12440. doi:10.1093/nar/gkx927 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Figure 6. Effect of calcium on exonuclease and DNA polymerase activities by various families of DNA Pols. The characteristics of families A, B, D, X and Y DNA Pols distributed across the three kingdom of life are summarized. The primer-template used for primer extension experiments and 3′-exonuclease primer degradation, in panels A–C and B–D, consists of a Cy5-labeled 17-mer primer annealed to a DNA template of 87-nt in length. (A–C) Extension of the 17/87 primer-template by DNA Pols at fixed Mg<sup>2+</sup> or Ca<sup>2+</sup> concentrations (5 mM). The extension (%) for selected lanes is shown under the gel. Reference oligodeoxynucleotides of 17 and 87 bases are indicated by the arrows. (B–D) Proofreading exonucleolysis of 17/87 primer-template at 5 mM Mg<sup>2+</sup> or Ca<sup>2+</sup> concentrations. The degradation (%) for selected lanes is shown under the gel. Reference oligodeoxynucleotides of 17 and 8 bases are indicated by the arrows. Black lines separate lanes which were not adjacent in the original gel. From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Nucleic Acids Res. 2017;45(21):12425-12440. doi:10.1093/nar/gkx927 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Figure 7. Schematic illustration of a model for the regulation of exonuclease and DNA polymerase catalyses by metal ions. (A) Diagram of the effects of Mg<sup>2+</sup> or/and Ca<sup>2+</sup> on PabPolB. The first two columns point out the kinetic parameters (K<sub>m</sub> and K<sub>cat</sub>) and the primer/template binding affinity (K<sub>D</sub> p/t) when PabPolB selects the correct versus incorrect dNTP. The third column shows the primer/template binding affinity (K<sub>D</sub> p/t) of PabPolB in exonuclease mode. The black triangles denote the drift constants from low to high. The two-color gradient in the background indicates the presence of Mg<sup>2+</sup> (blue) or Ca<sup>2+</sup> (green) alone, or a mixture of Mg<sup>2+</sup> and Ca<sup>2+</sup> (between blue and green). (B) Model of the modulation of DNA polymerase and exonuclease activities by fluctuating metal ion concentration in archaeal cells. PabPolB exonuclease and polymerase domains are light and dark blue with Mg<sup>2+</sup> or light and dark green with Ca<sup>2+</sup>. ‘mis’ denotes misinserted nucleotide which is represented by a red cross. The schematic illustration on the left describes the activation of DNA polymerase and exonuclease activities by Mg<sup>2+</sup>. The nucleotide incorporation cycle is accurate, fast and balanced by the excision of mismatches. At any time, PabPolB remains susceptible to changes in metal ion content in response to external stress (dashed rightward arrow over leftward arrow). The schematic illustration on the right shows the tuning effects (DNA polymerase activation and exonuclease inactivation) in the presence of Ca<sup>2+</sup>. PabPolB binds the p/t with higher affinity and DNA synthesis is slower but more accurate. When PabPolB is stalled by a mismatch in an exonuclease mode, displacement of Ca<sup>2+</sup> ions by local Mg<sup>2+</sup> spikes reactivates its proofreading function (dashed arrow). From: Calcium-driven DNA synthesis by a high-fidelity DNA polymerase Nucleic Acids Res. 2017;45(21):12425-12440. doi:10.1093/nar/gkx927 Nucleic Acids Res | © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com