Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, MI 48859, USA.
Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, MI 48859, USA; Biochemistry, Cellular and Molecular Biology Graduate Program, Central Michigan University, Mount Pleasant, MI 48859, USA.
J Mol Biol. 2019 Feb 15;431(4):673-686. doi: 10.1016/j.jmb.2019.01.002. Epub 2019 Jan 8.
PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn, a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas in the presence of Mg, the chemical step becomes rate limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase.
PrimPol 是最近发现的人类 DNA 聚合酶/引发酶，在核和线粒体基因组维持中发挥着新兴作用。作为古细菌-真核引发酶超家族酶的成员，PrimPol 具有 DNA 聚合酶和引发酶活性，对于体外和细胞内复制叉的进展非常重要。PrimPol 的酶活性严重依赖于核苷酸转移反应，以连续掺入脱氧核糖核苷酸；然而，我们对反应的动力学机制的了解仍然不完整。使用酶动力学分析和计算机模拟，我们剖析了 PrimPol 将核苷酸转移到引物-模板 DNA 的机制，该机制包括 DNA 结合、构象转变、核苷酸结合、磷酸二酯键形成和解离步骤。我们通过稳态和预稳态动力学分析和模拟获得了步骤的速率常数。我们的数据表明，PrimPol 催化的 DNA 延伸的限速步骤取决于涉及的金属辅因子。在 Mn 的存在下，从非生产性到生产性 PrimPol:DNA 复合物的构象转变步骤限制了酶的周转率，而在 Mg 的存在下，化学步骤成为限速步骤。正如我们的动力学和模拟数据所证明的那样，PrimPol 在毫摩尔或生理微摩尔 Mn 浓度下保持相同的动力学机制。我们的研究揭示了 PrimPol 用两种常见金属辅因子催化核苷酸掺入的基础机制，并为进一步理解这种功能多样的引发酶-聚合酶的调节机制提供了动力学基础。
