Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae
Abstract
:1. Introduction
2. Results and Discussion
2.1. Influence of Reaction Conditions on 3′–5′–Exonuclease Activity of Apn2
2.2. Accumulation Kinetics of Products of the 3′–5′–Exonuclease Reaction
2.3. Determining the Inhibitory Action of 2′–Deoxynucleoside Monophosphates on the 3′–5′–Exonuclease Activity of Apn2
2.4. Examination of Conformational Changes of Apn2 during the Enzymatic Cycle
2.5. Analysis of Conformational Changes in the DNA Substrate during the Enzymatic Cycle
3. Materials and Methods
3.1. DNA Substrates
3.2. The Enzyme
3.3. Electrophoresis in a Denaturing Polyacrylamide Gel (PAAG)
3.4. The Stopped–Flow Method
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
References
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DNA Substrate | Ks, μM−1 | kcat, ×103 s−1 |
---|---|---|
FAMExo–C/G | 0.40 ± 0.20 | 2.7 ± 1.2 |
FAMExo–G/C | 3.0 ± 0.3 | |
FAMExo–T/A | 3.3 ± 1.2 | |
FAMExo–A/T | 7.5 ± 0.7 | |
FAMExo–C/A | 13.3 ± 3.3 | |
FAMExo–C/T | 12.0 ± 0.2 | |
FAMExo–C/C | 16.7 ± 5.0 | |
FAMExo–CCC/GGG | 1.0 ± 1.2 | 2.4 ± 0.4 |
FAMExo–GGG/CCC | 0.3 ± 0.4 | 3.4 ± 1.6 |
FAMExo–TTT/AAA | 0.7 ± 0.4 | 2.2 ± 0.1 |
FAMExo–AAA/TTT | 7.2 ± 4.1 | 62 ± 25 |
Name | Sequence |
---|---|
FAMExo–X/Y, where X=G/A/T/C, Y=G/A/T/C | 5′–FAM–CAGCTCTGTACGTGAGX–3′ 3′–GTCGAGACATGCACTCYTCACCACTGTG–5′ |
TrpExo–G/C | 5′–CAGCTCTGTACGTGAGG–3′ 3′–GTCGAGACATGCACTCCTCACCACTGTG–5′ |
FAMExo–CCC/GGG | 5′–FAM–CAGCTCTGTACGTGCCC–3′ 3′–GTCGAGACATGCACGGGTCACCACTGTG–5′ |
FAMExo–GGG/CCC | 5′–FAM–CAGCTCTGTACGTGGGG–3′ 3′–GTCGAGACATGCACCCCTCACCACTGTG–5′ |
FAMExo–TTT/AAA | 5′–FAM–CAGCTCTGTACGTGTTT–3′ 3′–GTCGAGACATGCACAAATCACCACTGTG–5′ |
FAMExo–AAA/TTT | 5′–FAM–CAGCTCTGTACGTGAAA–3′ 3′–GTCGAGACATGCACTTTTCACCACTGTG–5′ |
Exo–aPu1 | 5′–CAGCTCTGTACGTGAGaPu–3′ 3′–GTCGAGACATGCACTC T TCACCACTGTG–5′ |
Exo–aPu2 | 5′–CAGCTCTGTACGTGAaPuT–3′ 3′–GTCGAGACATGCACT T ATCACCACTGTG–5′ |
Exo–aPuopposite | 5′–CAGCTCTGTACGTGAG T–3′ 3′–GTCGAGACATGCACTCaPuTCACCACTGTG–5′ |
Exo–1CPy | 5′–CAGCTCTGTACGTGAGCpy–3′ 3′–GTCGAGACATGCACTCGTCACCACTGTG–5′ |
Exo–2CPy | 5′–CAGCTCTGTACGTGACpyT–3′ 3′–GTCGAGACATGCACT G ATCACCACTGTG–5′ |
Exo–oppositeCPy | 5′–CAGCTCTGTACGTGAGG–3′ 3′–GTCGAGACATGCACTCCpyTCACCACTGTG–5′ |
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Kuznetsova, A.A.; Gavrilova, A.A.; Ishchenko, A.A.; Saparbaev, M.; Fedorova, O.S.; Kuznetsov, N.A. Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae. Int. J. Mol. Sci. 2022, 23, 14404. https://doi.org/10.3390/ijms232214404
Kuznetsova AA, Gavrilova AA, Ishchenko AA, Saparbaev M, Fedorova OS, Kuznetsov NA. Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae. International Journal of Molecular Sciences. 2022; 23(22):14404. https://doi.org/10.3390/ijms232214404
Chicago/Turabian StyleKuznetsova, Aleksandra A., Anastasia A. Gavrilova, Alexander A. Ishchenko, Murat Saparbaev, Olga S. Fedorova, and Nikita A. Kuznetsov. 2022. "Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae" International Journal of Molecular Sciences 23, no. 22: 14404. https://doi.org/10.3390/ijms232214404