Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, United States.
DNA Repair (Amst). 2013 Nov;12(11):922-35. doi: 10.1016/j.dnarep.2013.08.008. Epub 2013 Sep 10.
Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol δ/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase δ were studied: Pol δ4 and Pol δ3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5'-blocking oligonucleotide. Pol δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. While Pol δ3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol δ4 produces cleavage fragments of 1-10 nts at low Fen1 concentrations. Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol δ3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol δ in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol δ3 has a role in lagging strand synthesis, and that both forms of Pol δ may participate in DNA replication in higher eukaryotic cells.
滞后链 DNA 复制需要 DNA 聚合酶 δ、Fen1 和 DNA 连接酶 I 的协同作用，以在连接冈崎片段之前去除 RNA/DNA 引物。为了更好地理解人类细胞中的这一过程，我们使用纯化的人类蛋白和寡核苷酸底物在体外重新构建了通过短链通路的冈崎片段加工。我们系统地表征了冈崎片段加工中的关键事件：链置换、Pol δ/Fen1 联合反应以去除 RNA/DNA 引物，以及与 DNA 连接酶 I 的完整反应。研究了两种形式的人类 DNA 聚合酶 δ：Pol δ4 和 Pol δ3，它们分别代表异四聚体和缺少 p12 亚基的异三聚体。与 Pol δ4 相比，Pol δ3 的链置换活性非常有限，并且在遇到 5'阻断寡核苷酸时会停滞。Pol δ4 和 Pol δ3 在 Pol δ/Fen1 反应中表现出不同的特征。虽然 Pol δ3 产生的主要是 1 和 2 个核苷酸的切割产物，而与 Fen1 浓度无关，但 Pol δ4 在低 Fen1 浓度下产生 1-10 个核苷酸的切割片段。Pol δ3 和 Pol δ4 在完整系统中表现出相似的连接产物形成。虽然两者都能够在体外进行冈崎片段加工，但 Pol δ3 表现出理想的特征，适合在冈崎片段加工中发挥作用。Pol δ3 容易空闲，与 Fen1 结合后主要产生 1 个核苷酸的切割产物，因此在去除阻塞链时，缺口平移占主导地位，避免产生需要进一步加工的更长的链环。这些研究代表了对人类 Pol δ 的两种形式在冈崎片段加工中的首次分析。研究结果为新的概念提供了证据，即 Pol δ3 在滞后链合成中起作用，并且两种形式的 Pol δ 都可能参与真核细胞中的 DNA 复制。
