Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka-shi, Fukuoka, 812-8582, Japan.
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, Fukuoka, 819-0395, Japan.
BMC Biol. 2020 Oct 28;18(1):152. doi: 10.1186/s12915-020-00889-y.
DNA polymerase D (PolD) is the representative member of the D family of DNA polymerases. It is an archaea-specific DNA polymerase required for replication and unrelated to other known DNA polymerases. PolD consists of a heterodimer of two subunits, DP1 and DP2, which contain catalytic sites for 3'-5' editing exonuclease and DNA polymerase activities, respectively, with both proteins being mutually required for the full activities of each enzyme. However, the processivity of the replicase holoenzyme has additionally been shown to be enhanced by the clamp molecule proliferating cell nuclear antigen (PCNA), making it crucial to elucidate the interaction between PolD and PCNA on a structural level for a full understanding of its functional relevance. We present here the 3D structure of a PolD-PCNA-DNA complex from Thermococcus kodakarensis using single-particle cryo-electron microscopy (EM).
Two distinct forms of the PolD-PCNA-DNA complex were identified by 3D classification analysis. Fitting the reported crystal structures of truncated forms of DP1 and DP2 from Pyrococcus abyssi onto our EM map showed the 3D atomic structural model of PolD-PCNA-DNA. In addition to the canonical interaction between PCNA and PolD via PIP (PCNA-interacting protein)-box motif, we found a new contact point consisting of a glutamate residue at position 171 in a β-hairpin of PCNA, which mediates interactions with DP1 and DP2. The DNA synthesis activity of a mutant PolD with disruption of the E171-mediated PCNA interaction was not stimulated by PCNA in vitro.
Based on our analyses, we propose that glutamate residues at position 171 in each subunit of the PCNA homotrimer ring can function as hooks to lock PolD conformation on PCNA for conversion of its activity. This hook function of the clamp molecule may be conserved in the three domains of life.
DNA 聚合酶 D（PolD）是 DNA 聚合酶 D 家族的代表性成员。它是一种古菌特异性 DNA 聚合酶，是复制所必需的，与其他已知的 DNA 聚合酶无关。PolD 由两个亚基 DP1 和 DP2 组成的异二聚体组成，分别包含 3'-5'编辑外切核酸酶和 DNA 聚合酶活性的催化位点，这两种蛋白质相互需要才能充分发挥每种酶的活性。然而，复制酶全酶的持续性也被证明可以通过增殖细胞核抗原（PCNA）分子增强，因此在结构水平上阐明 PolD 与 PCNA 之间的相互作用对于充分了解其功能相关性至关重要。我们在这里使用单颗粒冷冻电子显微镜（EM）呈现了来自 Thermococcus kodakarensis 的 PolD-PCNA-DNA 复合物的 3D 结构。
通过 3D 分类分析，确定了两种不同形式的 PolD-PCNA-DNA 复合物。将来自 Pyrococcus abyssi 的 DP1 和 DP2 的截短形式的报告晶体结构拟合到我们的 EM 图谱上，显示了 PolD-PCNA-DNA 的 3D 原子结构模型。除了 PCNA 通过 PIP（PCNA 相互作用蛋白）盒基序与 PolD 的典型相互作用外，我们还发现了一个新的接触点，由 PCNA 中的一个β发夹中的谷氨酸残基 171 组成，该残基介导与 DP1 和 DP2 的相互作用。在体外，破坏 E171 介导的 PCNA 相互作用的突变 PolD 的 DNA 合成活性不能被 PCNA 刺激。
基于我们的分析，我们提出 PCNA 同源三聚体环中每个亚基的 171 位谷氨酸残基可以作为钩子，将 PolD 构象锁定在 PCNA 上，以转换其活性。夹钳分子的这种钩子功能可能在生命的三个领域中都得到了保守。
