School of Molecular Sciences, The Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
Nephrology and Hypertension Research, Division of Hypertension and Nephrology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA.
J Biol Chem. 2021 Aug;297(2):100912. doi: 10.1016/j.jbc.2021.100912. Epub 2021 Jun 24.
The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.
跨损伤合成（TLS）DNA 聚合酶 Rev1 和 Polζ 在 DNA 复制过程中共同作用于 DNA 损伤绕过，分别作为核苷酸插入酶和延伸酶聚合酶。虽然酿酒酵母 Polζ 在其 DNA 结合状态下的结构特征阐明了该酶如何合成 DNA，但对 TLS 的机制理解还需要探测与 DNA 和 Rev1 结合相关的构象变化。在这里，我们使用单颗粒冷冻电镜确定了apo Polζ 全酶的结构。我们表明，与 DNA 结合状态相比，apo Polζ 显示出增强的灵活性，这与 DNA 结合时 Polζ DNA 结合通道扩张相关的协调运动相关。我们还鉴定了一个在 apo Polζ 中阻碍 DNA 结合通道的赖氨酸残基，表明存在一种门控机制。Polζ 亚基 Rev7 是一种直接结合 Rev1 的枢纽蛋白，并且是几种其他蛋白质复合物（如shieldin DNA 双链断裂修复复合物）的组成部分。我们分析了酿酒酵母 Rev7 在 Polζ 中的分子相互作用以及人 Rev7 在 shieldin 中的分子相互作用，使用 Rev7 与 shieldin-3 蛋白片段结合的晶体结构。总体而言，我们的研究为 Polζ 的作用机制以及 Rev7 识别伴侣蛋白的方式提供了新的见解。
