Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Oligonucleotides
2.2. Expression and Purification of Recombinant Pa-PolDom
2.3. Site-Directed Mutagenesis
2.4. Nucleotide Insertion Assays on Defined DNA Molecules
2.5. Steady-State Primer Extension Assays
2.6. Electrophoretic Mobility Shift Assays (EMSAs)
2.7. Kinetic Measurement of 8oxoGMP Incorporation by PaPolDom
3. Results and Discussion
3.1. Role of Pa-LigD Residues His651 and Ser768 in Preferential Insertion of Ribonucleotides and Fidelity
3.2. Role of Pa-LigD Lys606 in Dislocation of Proximal Templating Nucleotides
3.3. 8oxoGMP Is Efficiently Inserted During In Vitro Polymerization by PolDom
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Appendix A
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Protein | Nucleotide | kcat (s−1) | Km (nM) | Cat.eff. (s−1·nM−1) | f | Fins |
---|---|---|---|---|---|---|
wild−type | UTP dTTP GTP | 26.9 ± 0.8 2.2 ± 0.1 0.92 ± 0.02 | 58.4 ± 5.1 33.3 ± 4.9 1211 ± 90 | 0.46 ± 0.02 0.07 ± 0.01 8 × 10−4 ± 3 × 10−5 | 7 | 625 |
S768Y | UTP dTTP GTP | 14.8 ± 0.4 0.42 ± 0.02 0.49 ± 0.01 | 55.5 ± 3.5 70.4 ± 9.9 865 ± 70 | 0.27 ± 0.01 6 × 10−3 ± 9 × 10−4 6 × 10−4 ± 3 × 10−5 | 44 | 473 |
S768A | UTP dTTP GTP | 22.6 ± 1.5 2.2 ± 0.1 0.25 ± 4 ×10−3 | 46 ± 9 31.2± 4.6 890 ± 66 | 0.49 ± 0.15 0.07 ± 3 × 10−3 3 × 10−4 ± 6 × 10−6 | 7 | 1749 |
H651A | UTP dTTP GTP | 6.2 ± 0.2 2.2 ± 0.2 0.02 ± 4 × 10−4 | 117 ± 10 60 ± 13 2385 ± 200 | 0.05 ± 4 × 10−3 0.04 ± 6 × 10−3 7 × 10−6 ± 7 ×1 0−7 | 1.3 | 7361 |
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Sánchez-Salvador, A.; de Vega, M. Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom. Biomolecules 2020, 10, 203. https://doi.org/10.3390/biom10020203
Sánchez-Salvador A, de Vega M. Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom. Biomolecules. 2020; 10(2):203. https://doi.org/10.3390/biom10020203
Chicago/Turabian StyleSánchez-Salvador, Alejandro, and Miguel de Vega. 2020. "Structural Determinants Responsible for the Preferential Insertion of Ribonucleotides by Bacterial NHEJ PolDom" Biomolecules 10, no. 2: 203. https://doi.org/10.3390/biom10020203