Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Methods. 2010 Jul;51(3):347-57. doi: 10.1016/j.ymeth.2010.02.017. Epub 2010 Feb 21.
Eukaryotic DNA replication is a complex process requiring the proper functioning of a multitude of proteins to create error-free daughter DNA strands and maintain genome integrity. Even though synthesis and joining of Okazaki fragments on the lagging strand involves only half the DNA in the nucleus, the complexity associated with processing these fragments is about twice that needed for leading strand synthesis. Flap endonuclease 1 (FEN1) is the central component of the Okazaki fragment maturation pathway. FEN1 cleaves flaps that are displaced by DNA polymerase delta (pol delta), to create a nick that is effectively joined by DNA ligase I. The Pif1 helicase and Dna2 helicase/nuclease contribute to the maturation process by elongating the flap displaced by pol delta. Though the reason for generating long flaps is still a matter of debate, genetic studies have shown that Dna2 and Pif1 are both important components of DNA replication. Our current knowledge of the exact enzymatic steps that govern Okazaki fragment maturation has heavily derived from reconstitution reactions in vitro, which have augmented genetic information, to yield current mechanistic models. In this review, we describe both the design of specific DNA substrates that simulate intermediates of fragment maturation and protocols for reconstituting partial and complete lagging strand replication.
真核生物 DNA 复制是一个复杂的过程，需要多种蛋白质的正常功能来产生无错误的子 DNA 链并维持基因组完整性。尽管滞后链上的 Okazaki 片段的合成和连接只涉及核内一半的 DNA，但与处理这些片段相关的复杂性大约是前导链合成所需的两倍。核酸内切酶 1（FEN1）是 Okazaki 片段成熟途径的核心组成部分。FEN1 切割由 DNA 聚合酶 δ（pol δ）置换的凸起，形成一个切口，有效地由 DNA 连接酶 I 连接。Pif1 解旋酶和 Dna2 解旋酶/核酸酶通过延伸 pol δ 置换的凸起来促进成熟过程。尽管产生长凸起的原因仍存在争议，但遗传研究表明 Dna2 和 Pif1 都是 DNA 复制的重要组成部分。我们目前对控制 Okazaki 片段成熟的确切酶促步骤的了解主要来自体外重建反应，这些反应增加了遗传信息，以产生当前的机制模型。在这篇综述中，我们描述了模拟片段成熟中间产物的特定 DNA 底物的设计以及部分和完整滞后链复制的重建方案。
