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

被引：16

作者：

Tokarsky, E. John ^{[1
,2
]}

Wallenmeyer, Petra C. ^{[1
]}

Phi, Kenneth K. ^{[1
]}

Suo, Zucai ^{[1
,2
]}

机构：

[1] Ohio State Univ, Dept Chem & Biochem, Columbus, OH 43210 USA

[2] Ohio State Univ, Ohio State Biophys Program, Columbus, OH 43210 USA

来源：

DNA REPAIR | 2017年 / 49卷

基金：

美国国家卫生研究院;

关键词：

Human PrimPol; Pre-steady-state kinetics; Enzyme regulation; Polymerase fidelity; DNA polymerase sugar selectivity; MITOCHONDRIAL-DNA POLYMERASE; SULFOLOBUS-SOLFATARICUS P2; NUCLEOTIDE ANALOGS; MANGANESE MUTAGENESIS; 1ST-LINE THERAPY; STRUCTURAL BASIS; KINETIC BASIS; POLYMERIZATION; REPLICATION; GEMCITABINE;

D O I：

10.1016/j.dnarep.2016.11.003

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

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 979 nM 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 x 10(-2) to 3.8 x 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). (C) 2016 Elsevier B.V. All rights reserved.

引用

页码：51 / 59

页数：9