title: Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol.
abstract: Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn2+-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn2+, rather than Mg2+, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn2+and Mg2+, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn2+than with Mg2+. Notably, the substitution fidelity of PrimPol in the presence of Mn2+was determined to be in the range of 3.4×10-2to 3.8×10-1, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn2+and 150-4500 with Mg2+, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biophysics Program, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: E John
authors.initials: EJ
authors.lastname: Tokarsky
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Petra C
authors.initials: PC
authors.lastname: Wallenmeyer
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Phi
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA; The Ohio State Biophysics Program, The Ohio State University, Columbus, OH 43210, USA. Electronic address: suo.3@osu.edu.
authors.forename: Zucai
authors.initials: Z
authors.lastname: Suo
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism / Q000627:therapeutic use; D000998:Antiviral Agents / Q000378:metabolism / Q000627:therapeutic use; D001085:Arabinofuranosylcytosine Triphosphate / Q000378:metabolism / Q000627:therapeutic use; D002413:Cations, Divalent / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism*; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000627:therapeutic use; D004247:DNA / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D054306:Dideoxynucleotides / Q000378:metabolism / Q000627:therapeutic use; D000068679:Emtricitabine / Q000031:analogs & derivatives / Q000378:metabolism / Q000627:therapeutic use; D006801:Humans; D007700:Kinetics; D019259:Lamivudine / Q000031:analogs & derivatives / Q000378:metabolism / Q000627:therapeutic use; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D064251:Multifunctional Enzymes / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; D000998:Antiviral Agents; D002413:Cations, Divalent; D003067:Coenzymes; D003854:Deoxyribonucleotides; D054306:Dideoxynucleotides; D064251:Multifunctional Enzymes; C417268:lamivudine triphosphate; D001085:Arabinofuranosylcytosine Triphosphate; D019259:Lamivudine; D008345:Manganese; D003570:Cytidine Triphosphate; D004247:DNA; C533693:gemcitabine triphosphate; D019915:DNA Primase; C585901:PrimPol protein, human; D004259:DNA-Directed DNA Polymerase; D000068679:Emtricitabine; D008274:Magnesium
keywords: DNA polymerase sugar selectivity; Enzyme regulation; Human PrimPol; Polymerase fidelity; Pre-steady-state kinetics