Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, United States.
Department of Biology, University of Iowa, Iowa City, IA, United States.
Methods Enzymol. 2022;672:339-368. doi: 10.1016/bs.mie.2022.02.025. Epub 2022 Mar 29.
Break-Induced Replication (BIR) is a homologous recombination (HR) pathway that differentiates itself from all other HR pathways by involving extensive DNA synthesis of up to hundreds of kilobases. This DNA synthesis occurs in G2/M arrested cells by a mechanism distinct from regular DNA replication. BIR initiates by strand invasion of a single end of a DNA double-strand break (DSB) followed by extensive D-loop migration. The main replicative helicase Mcm2-7 is dispensable for BIR, however, Pif1 helicase and its PCNA interaction domain are required. Pif1 helicase was shown to be important for extensive repair-specific DNA synthesis at DSB in budding and fission yeasts, flies, and human cells, implicating conservation of the mechanism. Additionally, Mph1 helicase negatively regulates BIR by unwinding migrating D-loops, and Srs2 promotes BIR by eliminating the toxic joint molecules. Here, we describe the methods that address the following questions in studying BIR: (i) how to distinguish enzymes needed specifically for BIR from enzymes needed for other HR mechanisms that require short patch DNA synthesis, (ii) what are the phenotypes expected for mutants deficient in extensive synthesis during BIR, (iii) how to follow extensive DNA synthesis during BIR? Methods are described using yeast model organism and wild-type cells are compared side-by-side with Pif1 deficient cells.
断裂诱导复制（BIR）是一种同源重组（HR）途径，通过涉及长达数百千碱基的广泛 DNA 合成，使其与所有其他 HR 途径区分开来。这种 DNA 合成发生在 G2/M 期停滞的细胞中，其机制与常规 DNA 复制不同。BIR 通过单链 DNA 双链断裂（DSB）的一个末端的链入侵起始，然后进行广泛的 D 环迁移。主要复制解旋酶 Mcm2-7 对于 BIR 是可有可无的，但是 Pif1 解旋酶及其 PCNA 相互作用结构域是必需的。已经表明 Pif1 解旋酶对于芽殖酵母和裂殖酵母、果蝇和人类细胞中 DSB 处的广泛修复特异性 DNA 合成很重要，这表明该机制是保守的。此外，Mph1 解旋酶通过解开迁移的 D 环负调控 BIR，而 Srs2 通过消除有毒的连接分子促进 BIR。在这里，我们描述了用于研究 BIR 的以下问题的方法：（i）如何区分专门用于 BIR 的酶与需要短补丁 DNA 合成的其他 HR 机制所需的酶，（ii）在 BIR 期间缺乏广泛合成时预期的突变体表型，（iii）如何在 BIR 期间跟踪广泛的 DNA 合成？使用酵母模式生物描述了方法，并将野生型细胞与 Pif1 缺陷细胞并排进行比较。
