Department of Medical Biochemistry and Biophysics, Umeå University, 90187 Umeå, Sweden.
Centro de Biologia Molecular Severo Ochoa, E-28049 Madrid, Spain.
Nucleic Acids Res. 2021 Feb 26;49(4):2179-2191. doi: 10.1093/nar/gkab049.
Replication forks often stall at damaged DNA. To overcome these obstructions and complete the DNA duplication in a timely fashion, replication can be restarted downstream of the DNA lesion. In mammalian cells, this repriming of replication can be achieved through the activities of primase and polymerase PrimPol. PrimPol is stimulated in DNA synthesis through interaction with PolDIP2, however the exact mechanism of this PolDIP2-dependent stimulation is still unclear. Here, we show that PrimPol uses a flexible loop to interact with the C-terminal ApaG-like domain of PolDIP2, and that this contact is essential for PrimPol's enhanced processivity. PolDIP2 increases primer-template and dNTP binding affinities of PrimPol, which concomitantly enhances its nucleotide incorporation efficiency. This stimulation is dependent on a unique arginine cluster in PolDIP2. Since the polymerase activity of PrimPol alone is very limited, this mechanism, where the affinity for dNTPs gets increased by PolDIP2 binding, might be critical for the in vivo function of PrimPol in tolerating DNA lesions at physiological nucleotide concentrations.
复制叉经常在受损的 DNA 处停滞不前。为了克服这些障碍并及时完成 DNA 复制，复制可以在 DNA 损伤下游重新启动。在哺乳动物细胞中，这种复制的重新启动可以通过引物酶和聚合酶 PrimPol 的活性来实现。PrimPol 通过与 PolDIP2 的相互作用在 DNA 合成中被激活，然而，这种 PolDIP2 依赖性激活的确切机制尚不清楚。在这里，我们表明 PrimPol 使用一个柔性环与 PolDIP2 的 C 端 ApaG 样结构域相互作用，并且这种接触对于 PrimPol 的增强的延伸性是必需的。PolDIP2 增加了 PrimPol 的引物-模板和 dNTP 结合亲和力，同时增强了其核苷酸掺入效率。这种刺激依赖于 PolDIP2 中的一个独特的精氨酸簇。由于 PrimPol 自身的聚合酶活性非常有限，因此这种机制（即 PolDIP2 结合增加了 dNTP 的亲和力）可能对于 PrimPol 在生理核苷酸浓度下耐受 DNA 损伤的体内功能至关重要。
