Institute of Molecular Genetics, National Research Center "Kurchatov Institute", Kurchatov sq. 2, 123182 Moscow, Russia.
Institute of Gene Biology, Russian Academy of Sciences, Vavilov 34/5, 119334 Moscow, Russia.
Nucleic Acids Res. 2023 Aug 11;51(14):7541-7551. doi: 10.1093/nar/gkad507.
Human PrimPol possesses DNA primase and DNA polymerase activities and restarts stalled replication forks protecting cells against DNA damage in nuclei and mitochondria. The zinc-binding motif (ZnFn) of the C-terminal domain (CTD) of PrimPol is required for DNA primase activity but the mechanism is not clear. In this work, we biochemically demonstrate that PrimPol initiates de novo DNA synthesis in cis-orientation, when the N-terminal catalytic domain (NTD) and the CTD of the same molecule cooperate for substrates binding and catalysis. The modeling studies revealed that PrimPol uses a similar mode of initiating NTP coordination as the human primase. The ZnFn motif residue Arg417 is required for binding the 5'-triphosphate group that stabilizes the PrimPol complex with a DNA template-primer. We found that the NTD alone is able to initiate DNA synthesis, and the CTD stimulates the primase activity of NTD. The regulatory role of the RPA-binding motif in the modulation of PrimPol binding to DNA is also demonstrated.
人 PrimPol 具有 DNA 引发酶和 DNA 聚合酶活性，可重新启动停滞的复制叉，保护细胞核和线粒体中的细胞免受 DNA 损伤。PrimPol 的 C 末端结构域（CTD）的锌结合基序（ZnFn）对于 DNA 引发酶活性是必需的，但机制尚不清楚。在这项工作中，我们通过生物化学手段证明，当同一分子的 N 末端催化结构域（NTD）和 CTD 合作进行底物结合和催化时，PrimPol 以顺式取向起始从头 DNA 合成。建模研究表明，PrimPol 采用与人类引发酶相似的起始 NTP 协调模式。ZnFn 基序残基 Arg417 与 5'-三磷酸基团结合，稳定 PrimPol 与 DNA 模板-引物的复合物。我们发现，NTD 本身能够起始 DNA 合成，而 CTD 则刺激 NTD 的引发酶活性。还证明了 RPA 结合基序在调节 PrimPol 与 DNA 结合中的调控作用。
