King Abdullah University of Science and Technology, Division of Biological and Environmental Science and Engineering, Thuwal 23955, Saudi Arabia.
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA.
Nucleic Acids Res. 2018 Apr 6;46(6):2956-2974. doi: 10.1093/nar/gky082.
RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.
RNA-DNA 杂交引物由低保真度 DNA 聚合酶 α 合成，以启动真核生物滞后链合成，在冈崎片段 (OF) 成熟过程中必须有效地去除，以完成 DNA 复制。在此过程中，每个 OF 引物被置换，并且产生的 5'-单链发夹由结构特异性 5'-核酸内切酶，主要是 Flap Endonuclease 1 (FEN1) 切割，以产生可连接的切口。至少提出了两种模型来描述引物的去除，即短发夹途径和长发夹途径，分别涉及 FEN1 或 FEN1 以及复制蛋白 A (RPA) 和 Dna2 解旋酶/核酸酶。我们通过研究 FEN1 对短发夹和长发夹 DNA 底物的作用的动力学机制来解决途径选择的问题。使用单分子 FRET 和快速猝灭流批量切割测定法，我们表明，与短发夹底物不同，短发夹底物在 FEN1 与 DNA 之间的首次相遇中被结合、弯曲和切割，长发夹底物甚至在 DNA 结合和弯曲后也可以逃避切割。值得注意的是，即使在存在 RPA 的情况下，FEN1 也可以访问这两种底物，但是长发夹 DNA 的弯曲和切割会受到特异性抑制。我们提出，FEN1 试图处理短发夹和长发夹，但后者偶尔会错过切割，从而允许 RPA 结合并触发长发夹 OF 成熟途径。
