Edited by Jack W. Szostak, Massachusetts General Hospital, Boston, MA, and approved December 22, 2017 (received for review July 8, 2017)
All extant cellular organisms are thought to replicate their genomes using triphosphorylated substrates (dNTPs). Because only the α-phosphate is retained in the DNA backbone, both dNTPs and diphosphates (dNDPs) can in principle drive DNA synthesis. The activation barrier for the transphosphorylation is expected to be higher for dNDPs than for dNTPs, rendering the dNDP reactions slower; however, at elevated temperatures this penalty may be less prohibitive. We demonstrate DNA synthesis from dNDPs for a number of DNA polymerases, including bacterial and archaeal replicative and repair enzymes. Activation energy analysis of the forward (DNA synthesis) and reverse (phosphorolysis of DNA) reactions catalyzed by the Taq DNA polymerase shows that DNA synthesis is strongly favored, allowing surprisingly robust replication from low-energy substrates.
The activity of DNA polymerase underlies numerous biotechnologies, cell division, and therapeutics, yet the enzyme remains incompletely understood. We demonstrate that both thermostable and mesophilic DNA polymerases readily utilize deoxyribonucleoside diphosphates (dNDPs) for DNA synthesis and inorganic phosphate for the reverse reaction, that is, phosphorolysis of DNA. For Taq DNA polymerase, the KMs of the dNDP and phosphate substrates are ∼20 and 200 times higher than for dNTP and pyrophosphate, respectively. DNA synthesis from dNDPs is about 17 times slower than from dNTPs, and DNA phosphorolysis about 200 times less efficient than pyrophosphorolysis. Such parameters allow DNA replication without requiring coupled metabolism to sequester the phosphate products, which consequently do not pose a threat to genome stability. This mechanism contrasts with DNA synthesis from dNTPs, which yield high-energy pyrophosphates that have to be hydrolyzed to phosphates to prevent the reverse reaction. Because the last common ancestor was likely a thermophile, dNDPs are plausible substrates for genome replication on early Earth and may represent metabolic intermediates later replaced by the higher-energy triphosphates.
↵1Present address: Molecular Engineering and Sciences Institute, Department of Chemical Engineering, University of Washington, Seattle, WA 98195.
Author contributions: C.R.B. and A.L. designed research, performed research, analyzed data, and wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1712193115/-/DCSupplemental.
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