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.

中文翻译：

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DNA聚合酶D-PCNA-DNA的两种构型，一种古细菌复制体复合物，通过冷冻电子显微镜观察到

DNA聚合酶D（PolD）是DNA聚合酶D家族的代表成员。它是复制所需的古细菌特异性DNA聚合酶，与其他已知的DNA聚合酶无关。PolD由两个亚基的异二聚体DP1和DP2组成，它们分别具有3'-5'编辑外切核酸酶和DNA聚合酶活性的催化位点，两种蛋白是每种酶的全部活性所必需的。但是，另外还显示，夹钳增殖细胞核抗原（PCNA）可增强复制酶全酶的生产力，因此在结构水平上阐明PolD与PCNA之间的相互作用对于全面了解其功能相关性至关重要。我们在这里使用单粒子冷冻电子显微镜（EM），从热球菌Kodakarensis的PolD-PCNA-DNA复合物的3D结构。通过3D分类分析确定了PolD-PCNA-DNA复合体的两种不同形式。将据报道的深渊热球菌DP1和DP2的截短形式的晶体结构拟合到我们的EM图上显示了PolD-PCNA-DNA的3D原子结构模型。除了通过PIP（PCNA相互作用蛋白）盒基序在PCNA和PolD之间进行规范的相互作用外，我们还发现了一个新的接触点，该接触点由PCNA的β-发夹中171位的谷氨酸残基组成，该位点介导与DP1和CD1 DP2。体外PCNA不会刺激E171介导的PCNA相互作用破坏的突变PolD的DNA合成活性。根据我们的分析，我们提出，PCNA同三聚体环的每个亚基中171位的谷氨酸残基可以起钩子的作用，以锁定PCNA上PolD构象以转化其活性。钳分子的这种钩功能可以在生命的三个域中保守。