Molecular Dissection of the Primase and Polymerase Activities of Deep-Sea Phage NrS-1 Primase-Polymerase.

PrimPols are a class of primases that belong to the archaeo-eukaryotic primase (AEP) superfamily but have both primase and DNA polymerase activities. Replicative polymerase from NrS-1 phage (NrSPol) is a representative of the PrimPols. In this study, we identified key residues for the catalytic activity of NrSPol and found that a loop in NrSPol functionally replaces the zinc finger motif that is commonly found in other AEP family proteins. A helix bundle domain (HBD), conserved in the AEP superfamily, was recently reported to bind to the primase recognition site and to be crucial for initiation of primer synthesis. We found that NrSPol can recognize different primase recognition sites, and that the initiation site for primer synthesis is not stringent, suggesting that the HBD conformation is flexible. More importantly, we found that although the HBD-inactivating mutation impairs the primase activity of NrSPol, it significantly enhances the DNA polymerase activity, indicating that the HBD hinders the DNA polymerase activity. The conflict between the primase activity and the DNA polymerase activity in a single protein with the same catalytic domain may be one reason for why DNA polymerases are generally unable to synthesize DNA de novo.

PrimPols是一类引发酶，属于古菌 - 真核生物引发酶（AEP）超家族，但同时具有引发酶和DNA聚合酶活性。来自NrS - 1噬菌体的复制性聚合酶（NrSPol）是PrimPols的一个代表。在这项研究中，我们确定了NrSPol催化活性的关键残基，并发现NrSPol中的一个环在功能上替代了在其他AEP家族蛋白中常见的锌指结构域。AEP超家族中保守的螺旋束结构域（HBD）近期被报道可结合引发酶识别位点，并且对引物合成的起始至关重要。我们发现NrSPol能够识别不同的引发酶识别位点，并且引物合成的起始位点并不严格，这表明HBD的构象是灵活的。更重要的是，我们发现尽管使HBD失活的突变会损害NrSPol的引发酶活性，但它却显著增强了DNA聚合酶活性，这表明HBD阻碍了DNA聚合酶活性。在具有相同催化结构域的单一蛋白质中引发酶活性和DNA聚合酶活性之间的冲突可能是DNA聚合酶通常无法从头合成DNA的原因之一。