Department of Chemistry and Biotechnology, Tallinn University of Technology, Tallinn, 12618, Estonia.
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, 10016, USA.
Nat Commun. 2022 Nov 19;13(1):7099. doi: 10.1038/s41467-022-34911-4.
DNA polymerase epsilon (PolE) in an enzyme essential for DNA replication. Deficiencies and mutations in PolE cause severe developmental abnormalities and cancers. Paradoxically, the catalytic domain of yeast PolE catalytic subunit is dispensable for survival, and its non-catalytic essential function is linked with replicative helicase (CMG) assembly. Less is known about the PolE role in replication initiation in human cells. Here we use an auxin-inducible degron system to study the effect of POLE1 depletion on replication initiation in U2OS cells. POLE1-depleted cells were able to assemble CMG helicase and initiate DNA synthesis that failed shortly after. Expression of POLE1 non-catalytic domain rescued this defect resulting in slow, but continuous DNA synthesis. We propose a model where in human U2OS cells POLE1/POLE2 are dispensable for CMG assembly, but essential during later steps of replication initiation. Our study provides some insights into the role of PolE in replication initiation in human cells.
DNA 聚合酶 ε（PolE）是一种酶，对于 DNA 复制是必不可少的。PolE 的缺乏和突变会导致严重的发育异常和癌症。矛盾的是，酵母 PolE 催化亚基的催化结构域对于生存是可有可无的，其非催化的必需功能与复制解旋酶（CMG）组装有关。关于 PolE 在人细胞中复制起始的作用知之甚少。在这里，我们使用一种生长素诱导的降解结构域系统来研究 POLE1 缺失对 U2OS 细胞复制起始的影响。POLE1 耗尽的细胞能够组装 CMG 解旋酶并启动 DNA 合成，但在短时间后失败。POLE1 非催化结构域的表达挽救了这一缺陷，导致缓慢但持续的 DNA 合成。我们提出了一个模型，即在人 U2OS 细胞中，POLE1/POLE2 对于 CMG 组装不是必需的，但在复制起始的后期步骤中是必需的。我们的研究为 PolE 在人细胞复制起始中的作用提供了一些见解。
