Optimization of the expression, purification and polymerase activity reaction conditions of recombinant human PrimPol.

Elizaveta O. Boldinova,Gorazd Stojkovič,Sjoerd Wanrooij,Alena V. Makarova,Rafil Khairullin,Giovanni Maga

doi:10.1371/journal.pone.0184489

Abstract

Human PrimPol is a DNA primase/polymerase involved in DNA damage tolerance and prevents nuclear genome instability. PrimPol is also localized to the mitochondria, but its precise function in mitochondrial DNA maintenance has remained elusive. PrimPol works both as a translesion (TLS) polymerase and as the primase that restarts DNA replication after a lesion. However, the observed biochemical activities of PrimPol vary considerably between studies as a result of different reaction conditions used. To reveal the effects of reaction composition on PrimPol DNA polymerase activity, we tested the polymerase activity in the presence of various buffer agents, salt concentrations, pH values and metal cofactors. Additionally, the enzyme stability was analyzed under various conditions. We demonstrate that the reaction buffer with pH 6–6.5, low salt concentrations and 3 mM Mg2+ or 0.3–3 mM Mn2+ cofactor ions supports the highest DNA polymerase activity of human PrimPol in vitro. The DNA polymerase activity of PrimPol was found to be stable after multiple freeze-thaw cycles and prolonged protein incubation on ice. However, rapid heat-inactivation of the enzyme was observed at 37ºC. We also for the first time describe the purification of human PrimPol from a human cell line and compare the benefits of this approach to the expression in Escherichia coli and in Saccharomyces cerevisiae cells. Our results show that active PrimPol can be purified from E. coli and human suspension cell line in high quantities and that the activity of the purified enzyme is similar in both expression systems. Conversely, the yield of full-length protein expressed in S. cerevisiae was considerably lower and this system is therefore not recommended for expression of full-length recombinant human PrimPol.

Highlights

PrimPol is a unique DNA polymerase with primase activity [1,2,3]
Human PrimPol is a 560 a. a. protein encoded by the CCDC111 (PRIMPOL) gene and belongs to the archaeo-eukaryotic primase (AEP) superfamily [4]
Human cells contain a variety of DNA polymerases that differ in their functions and fidelity of DNA synthesis

Summary

Introduction

PrimPol is a unique DNA polymerase with primase activity [1,2,3]. Human PrimPol works as a translesion DNA polymerase and can bypass DNA lesions such as 8-oxo-G and pyrimidine-photoproducts [1, 2, 6, 7]. To other translesion DNA polymerases, PrimPol lacks the 3’-5’-exonuclease activity and exhibits low fidelity of DNA synthesis [6, 8]. PrimPol is only the second primase found in human cells along with the Pol α-primase complex. Unlike Pol α-primase PrimPol can synthesize primers using deoxyribonucleotides during de novo DNA synthesis [1, 2]. PrimPol can utilize both Mg2+ and Mn2+ ions as cofactors for both priming and polymerase activities [2, 6]

Methods

Results

Discussion

Conclusion