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DNA synthesis from diphosphate substrates by DNA polymerases.

Cassandra R Burke1, Andrej Lupták2,3,4

  • 1Department of Chemistry, University of California, Irvine, CA 92697.

Proceedings of the National Academy of Sciences of the United States of America
|January 18, 2018
PubMed
Summary

DNA polymerases can use deoxyribonucleoside diphosphates (dNDPs) for DNA synthesis and inorganic phosphate for DNA breakdown. This finding impacts our understanding of DNA replication and early life evolution.

Keywords:
DNA replicationactivation energyenergy chargephosphorolysistransition state

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Evolutionary Biology

Background:

  • DNA polymerase activity is crucial for biotechnology, cell division, and therapeutics but remains incompletely understood.
  • The precise substrates and reaction mechanisms of DNA polymerases are key to their function and regulation.

Purpose of the Study:

  • To investigate the utilization of deoxyribonucleoside diphosphates (dNDPs) and inorganic phosphate by DNA polymerases.
  • To compare the kinetics and efficiency of DNA synthesis and phosphorolysis using dNDPs versus deoxyribonucleoside triphosphates (dNTPs).

Main Methods:

  • Enzymatic assays were performed using both thermostable and mesophilic DNA polymerases.
  • Kinetic parameters (K_M) for dNDP, phosphate, dNTP, and pyrophosphate substrates were determined.
  • Reaction rates for DNA synthesis and phosphorolysis were measured and compared.

Main Results:

  • Both thermostable and mesophilic DNA polymerases efficiently utilize dNDPs for DNA synthesis and inorganic phosphate for DNA phosphorolysis.
  • Taq DNA polymerase exhibits significantly higher K_M values for dNDP and phosphate substrates compared to dNTP and pyrophosphate.
  • DNA synthesis from dNDPs is slower, and DNA phosphorolysis is less efficient than reactions involving dNTPs and pyrophosphates.

Conclusions:

  • The use of dNDPs and inorganic phosphate by DNA polymerases allows for DNA replication without immediate sequestration of phosphate products, preserving genome stability.
  • This mechanism contrasts with dNTP-based synthesis, which requires pyrophosphate hydrolysis to prevent the reverse reaction.
  • dNDPs are plausible substrates for early Earth genome replication, potentially predating the evolution of higher-energy dNTPs.