Poster abstracts

Poster number 69 submitted by Jack Tokarsky

Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol

Jack Tokarsky (Biophysics Graduate Program), Petra Wallenmeyer (Department of Chemistry and Biochemistry), Kenny Phi (Department of Chemistry and Biochemistry), Zucai Suo (Department of Chemistry and Biochemistry)

Abstract:
Human PrimPol is a recently discovered bifunctional enzyme that displays template-directed primase activity and DNA polymerase activity. 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 coordinating Mn2+, rather than Mg2+, as a divalent metal ion cofactor. However, the abundance of cellular Mg2+ is much greater than that of Mn2+. At present, there is debate among those who study PrimPol as to which divalent metal ion cofactor is utilized by this unique protein. We experimentally studied PrimPol in the presence of both cofactors in an attempt to elucidate which of them is preferred. Our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with the affinities of 29 and 979 nM in the presence of Mn2+ and Mg2+, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct nucleotides with 100-fold higher efficiency with Mn2+ than with Mg2+. Strikingly, the substitution fidelity of PrimPol in the presence of Mn2+ was determined to be in the range of 3.4x10-2 to 3.8x10-1, indicating that PrimPol displays extremely unfaithful polymerase activity, which may not be biologically relevant. Additionally, we demonstrated a simple kinetic basis for discrimination by PrimPol to select for deoxynucleotides over ribonucleotides with both Mg2+ and Mn2+ likely arising from decreased nucleotide binding and subsequent incorporation. Furthermore, PrimPol also showed efficient incorporation of the anti-cancer drugs, cytarabine and gemcitabine, onto growing DNA strands revealing its potential role in cellular toxicities induced by these drugs.

Keywords: PrimPol, Pre-Steady-State Kinetics