Waga S, Masuda T, Takisawa H, Sugino A
Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Proc Natl Acad Sci U S A. 2001 Apr 24;98(9):4978-83. doi: 10.1073/pnas.081088798. Epub 2001 Apr 10.
DNA polymerase epsilon (Pol epsilon) is thought to be involved in DNA replication, repair, and cell-cycle checkpoint control in eukaryotic cells. Although the requirement of other replicative DNA polymerases, DNA polymerases alpha and delta (Pol alpha and delta), for chromosomal DNA replication has been well documented by genetic and biochemical studies, the precise role, if any, of Pol epsilon in chromosomal DNA replication is still obscure. Here we show, with the use of a cell-free replication system with Xenopus egg extracts, that Xenopus Pol epsilon is indeed required for chromosomal DNA replication. In Pol epsilon-depleted extracts, the elongation step of chromosomal DNA replication is markedly impaired, resulting in significant reduction of the overall DNA synthesis as well as accumulation of small replication intermediates. Moreover, despite the decreased DNA synthesis, excess amounts of Pol alpha are loaded onto the chromatin template in Pol epsilon-depleted extracts, indicative of the failure of proper assembly of DNA synthesis machinery at the fork. These findings strongly suggest that Pol epsilon, along with Pol alpha and Pol delta, is necessary for coordinated chromosomal DNA replication in eukaryotic cells.
DNA聚合酶ε（Pol ε）被认为参与真核细胞中的DNA复制、修复及细胞周期检查点控制。尽管遗传和生化研究已充分证明其他复制性DNA聚合酶，即DNA聚合酶α和δ（Pol α和Pol δ）对染色体DNA复制的必要性，但Pol ε在染色体DNA复制中的确切作用（如果有）仍不清楚。在此我们利用非洲爪蟾卵提取物的无细胞复制系统表明，非洲爪蟾Pol ε确实是染色体DNA复制所必需的。在去除Pol ε的提取物中，染色体DNA复制的延伸步骤明显受损，导致总体DNA合成显著减少以及小复制中间体积累。此外，尽管DNA合成减少，但在去除Pol ε的提取物中，过量的Pol α被加载到染色质模板上，这表明在复制叉处DNA合成机器未能正确组装。这些发现有力地表明，Pol ε与Pol α和Pol δ一起，是真核细胞中协调染色体DNA复制所必需的。
