Kinetic Analysis of Human PrimPol DNA Polymerase Activity Reveals a Generally Error-Prone Enzyme Capable of Accurately Bypassing

ABSTRACT: Recent studies have identified human PrimPol as a new RNA/DNA primase and translesion DNA synthesis polymerase (TLS pol) that contributes to nuclear and mitochondrial DNA replication. We investigated the mecha-nism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg2+ as a cofactor, PrimPol binds primer-template DNA with low affinity Kd,DNA values (∼200−1200 nM). DNA binding is enhanced 34-fold by Mn2+ (Kd,DNA = 27 nM). The pol activity of PrimPol is increased 400−1000-fold by Mn2+ compared to Mg2+ based on steady-state kinetic parameters. PrimPol makes a mistake copying undamaged DNA once every ∼100−2500 insertions events, which is comparable to other TLS pols, and the fidelity of PrimPol is ∼1.7-fold more accurate when Mg2+ is the cofactor compared to Mn2+. PrimPol inserts dCMP opposite 8-oxo-dG with 2- (Mn2+) to 6-fold (Mg2+) greater efficiency than dAMP misinsertion. PrimPol-catalyzed dCMP insertion opposite 8-oxo-dG proceeds at ∼25 % efficiency relative to unmodified template dG, and PrimPol readily extends from dC:8-oxo-dG base pairs (bps) with ∼2-fold greater efficiency than dA:8-oxo-dG bps. A tetrahydrofuran (THF) abasic-site mimic decreases PrimPol activity to ∼0.04%