Substrate energetics in non-templated DNA synthesis by terminal deoxynucleotidyl transferase
- 1Department of Pharmaceutical Sciences, University of California , Irvine, CA 92697 ,
- 2Department of Chemistry, University of California , Irvine, CA 92697 ,
- 3Department of Molecular Biology and Biochemistry, University of California , Irvine, CA 92697 ,
Abstract
DNA polymerases typically use deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis, yet the contribution of the γ-phosphate to catalysis remains unclear. Here, we examine the role of substrate energetics in enzyme-catalyzed polymerization using a fidelity-independent model polymerase, terminal deoxynucleotidyl transferase (TdT). Relative to dNTPs, we find that TdT utilizes the canonical deoxyribonucleoside diphosphates (dNDPs) with reduced rates and catalytic efficiencies, but diminished substrate bias. This lower specificity indicates that the γ-phosphate contributes primarily to nucleotide discrimination, rather than phosphodiester bond formation. Furthermore, our work shows that dNDPs and dNTPs with the same nucleobase exhibit comparable activation energies for the rate-limiting step during DNA synthesis, supporting a model in which the γ-phosphate is not required for lowering the energetic barrier for TdT-catalyzed polymerization. These findings define a mechanistic framework for understanding non-templated DNA synthesis and its application in enzymatic DNA synthesis technologies.
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BibTeX
@article{mogannam2026substrate,
title = {{Substrate energetics in non-templated DNA synthesis by terminal deoxynucleotidyl transferase}},
author = {Katrina Mogannam and Andrej Lupták},
journal = {Nucleic Acids Research},
year = {2026},
month = oct,
doi = {10.1093/nar/gkag980},
url = {https://academic.oup.com/nar/article/doi/10.1093/nar/gkag980/8915330},
note = {CC BY-NC 4.0},
}
RIS
TY - JOUR
TI - Substrate energetics in non-templated DNA synthesis by terminal deoxynucleotidyl transferase
AU - Katrina Mogannam
AU - Andrej Lupták
PY - 2026
DA - 2026/10/14
JO - Nucleic Acids Research
DO - 10.1093/nar/gkag980
UR - https://academic.oup.com/nar/article/doi/10.1093/nar/gkag980/8915330
AB - DNA polymerases typically use deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis, yet the contribution of the γ-phosphate to catalysis remains unclear. Here, we examine the role of substrate energetics in enzyme-catalyzed polymerization using a fidelity-independent model polymerase, terminal deoxynucleotidyl transferase (TdT). Relative to dNTPs, we find that TdT utilizes the canonical deoxyribonucleoside diphosphates (dNDPs) with reduced rates and catalytic efficiencies, but diminished substrate bias. This lower specificity indicates that the γ-phosphate contributes primarily to nucleotide discrimination, rather than phosphodiester bond formation. Furthermore, our work shows that dNDPs and dNTPs with the same nucleobase exhibit comparable activation energies for the rate-limiting step during DNA synthesis, supporting a model in which the γ-phosphate is not required for lowering the energetic barrier for TdT-catalyzed polymerization. These findings define a mechanistic framework for understanding non-templated DNA synthesis and its application in enzymatic DNA synthesis technologies.
SN - 0305-1048
SN - 1362-4962
ER -