Human primase and DNA polymerase PrimPol re-starts stalled replication forks by repriming downstream DNA lesions and protects cells against DNA damage. Structure of the catalytic core of PrimPol with DNA primer, template and incoming dATP was solved but the mechanisms of DNA polymerase and primase activities of PrimPol are not fully understood. In this work, using site-directed mutagenesis we biochemically analyzed the role of active site residues Arg47 and Arg76 contacting DNA template in DNA polymerase and primase activities of PrimPol. The substitution R47A diminished the DNA polymerase and primase activities of PrimPol whereas the single amino acid substitution R76A caused almost complete loss of catalytic activities. Both amino acid substitutions affected the spectrum of dNMPs incorporation on undamaged DNA templates and opposite 8-oxoguanine. Finally, substitutions of the Arg47 and Arg76 residues attenuated the formation of the stable PrimPol:DNA complex in the presence of ATP/dNTPs. Together, these findings suggest a key role of the Arg47 and Arg76 in DNA synthesis by PrimPol.

人类引发酶和 DNA 聚合酶 PrimPol 通过重新引发下游 DNA 损伤来重新启动停滞的复制叉，并保护细胞免受 DNA 损伤。PrimPol 催化核心与 DNA 引物、模板和传入 dATP 的结构已经解决，但 PrimPol 的 DNA 聚合酶和引发酶活性的机制尚不完全清楚。在这项工作中，我们使用定点诱变生化分析了接触 DNA 模板的活性位点残基 Arg47 和 Arg76 在 PrimPol 的 DNA 聚合酶和引物酶活性中的作用。替换 R47A 降低了 PrimPol 的 DNA 聚合酶和引物酶活性，而单个氨基酸替换 R76A 导致催化活性几乎完全丧失。两种氨基酸替换都会影响未损坏的 DNA 模板和相反的 8-氧鸟嘌呤上的 dNMP 掺入范围。最后，在 ATP/dNTP 存在下，Arg47 和 Arg76 残基的置换减弱了稳定的 PrimPol:DNA 复合物的形成。总之，这些发现表明 Arg47 和 Arg76 在 PrimPol 的 DNA 合成中起关键作用。