title: Kinetic analysis of human PrimPol DNA polymerase activity reveals a generally error-prone enzyme capable of accurately bypassing 7,8-dihydro-8-oxo-2'-deoxyguanosine.
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 mechanism of PrimPol polymerase activity on both undamaged and damaged DNA substrates. With Mg²⁺ as a cofactor, PrimPol binds primer-template DNA with low affinity K(d,DNA) values (∼200-1200 nM). DNA binding is enhanced 34-fold by Mn²⁺ (K(d,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%. In summary, PrimPol exhibits the fidelity typical of other TLS pols, is rather unusual in the degree of activation afforded by Mn²⁺, and accurately bypasses 8-oxo-dG, a DNA lesion of special relevance to mitochondrial DNA replication and transcription.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences , Little Rock, Arkansas 72205-7199, United States.
authors.forename: Maroof K
authors.initials: MK
authors.lastname: Zafar
authors.forename: Amit
authors.initials: A
authors.lastname: Ketkar
authors.forename: Maria F
authors.initials: MF
authors.lastname: Lodeiro
authors.forename: Craig E
authors.initials: CE
authors.lastname: Cameron
authors.forename: Robert L
authors.initials: RL
authors.lastname: Eoff
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D055162:Biocatalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D019915:DNA Primase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism*; D008958:Models, Molecular; D064251:Multifunctional Enzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic; D015854:Up-Regulation
chemical_list: D017931:DNA Primers; D064251:Multifunctional Enzymes; D011994:Recombinant Proteins; D008345:Manganese; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D019915:DNA Primase; C585901:PrimPol protein, human; D004259:DNA-Directed DNA Polymerase; D003849:Deoxyguanosine; D008274:Magnesium