Centro de Biología Molecular Severo Ochoa (CSIC-UAM), c/Nicolás Cabrera 1, 28049, Cantoblanco, Madrid, Spain.
Sci Rep. 2017 Apr 10;7(1):783. doi: 10.1038/s41598-017-00958-3.
Human PrimPol is a primase belonging to the AEP superfamily with the unique ability to synthesize DNA primers de novo, and a non-processive DNA polymerase able to bypass certain DNA lesions. PrimPol facilitates both mitochondrial and nuclear replication fork progression either acting as a conventional TLS polymerase, or repriming downstream of blocking lesions. In vivo assays have shown that PrimPol is rapidly recruited to sites of DNA damage by interaction with the human replication protein A (RPA). In agreement with previous findings, we show here that the higher affinity of RPA for ssDNA inhibits PrimPol activities in short ssDNA templates. In contrast, once the amount of ssDNA increases up to a length in which both proteins can simultaneously bind ssDNA, as expected during replicative stress conditions, PrimPol and RPA functionally interact, and their binding capacities are mutually enhanced. When using M13 ssDNA as template, RPA stimulated both the primase and polymerase activities of PrimPol, either alone or in synergy with Polε. These new findings supports the existence of a functional PrimPol/RPA association that allows repriming at the exposed ssDNA regions formed in the leading strand upon replicase stalling.
人源 PrimPol 是一种属于 AEP 超家族的引物酶，具有从头合成 DNA 引物的独特能力，是一种非连续的 DNA 聚合酶，能够绕过某些 DNA 损伤。PrimPol 能够作为传统的 TLS 聚合酶促进线粒体和核复制叉的延伸，或者在阻滞损伤下游重新引发引物。体内实验表明，PrimPol 通过与人类复制蛋白 A（RPA）相互作用，迅速被招募到 DNA 损伤部位。与先前的发现一致，我们在这里表明，RPA 对 ssDNA 的更高亲和力抑制了短 ssDNA 模板中 PrimPol 的活性。相比之下，一旦 ssDNA 的量增加到两种蛋白质都可以同时结合 ssDNA 的长度，就像在复制应激条件下所预期的那样，PrimPol 和 RPA 会发生功能相互作用，并且它们的结合能力会相互增强。当使用 M13 ssDNA 作为模板时，RPA 单独或与 Polε 协同刺激 PrimPol 的引物酶和聚合酶活性。这些新发现支持存在功能上的 PrimPol/RPA 关联，该关联允许在复制酶停滞时在先导链上形成的暴露 ssDNA 区域重新引发引物。
