Liu Jie, Sneeden Jessica, Heyer Wolf-Dietrich
Department of Microbiology, University of California, Davis, CA 95616-8665, USA.
Methods Mol Biol. 2011;745:363-83. doi: 10.1007/978-1-61779-129-1_21.
Homologous recombination (HR) is a high-fidelity DNA repair pathway that maintains genome integrity, by repairing double strand breaks (DSBs) and single-stranded DNA (ssDNA) gaps and by supporting stalled/collapsed replication forks. The RecA/Rad51 family of proteins are the key enzymes in this homology-directed repair pathway, as they perform DNA strand invasion and exchange, in concert with a host of ancillary factors. In vitro, the RecA/Rad51 family of proteins share similar enzymatic activities including catalyzing ssDNA-stimulated ATP hydrolysis, formation of displacement loops (D-loops), and DNA strand exchange. After successful DNA strand invasion, DNA synthesis restores the lost genetic information using an undamaged DNA template. In this chapter, we describe two well-established biochemical assays to investigate the signature DNA strand transfer activity of RecA/Rad51 family of proteins: the D-loop assay and the DNA strand exchange reaction. Moreover, we describe a D-loop extension assay coupling D-loop formation with DNA synthesis, which can be used to define the roles of DNA polymerases in HR. Additionally, we present a protocol to investigate protein-mediated DNA annealing, a critical step in the synthesis-dependent strand annealing (SDSA) and double-Holliday junction (dHJ) pathways as well as the single-strand annealing (SSA) pathway. The quality of supercoiled plasmid DNA is critical in reconstituted HR reactions, and a protocol describing the preparation of this DNA substrate is included.
同源重组（HR）是一种高保真的DNA修复途径，通过修复双链断裂（DSB）和单链DNA（ssDNA）缺口以及支持停滞/崩溃的复制叉来维持基因组完整性。RecA/Rad51家族蛋白是这种同源性定向修复途径中的关键酶，因为它们与许多辅助因子协同进行DNA链侵入和交换。在体外，RecA/Rad51家族蛋白具有相似的酶活性，包括催化ssDNA刺激的ATP水解、置换环（D环）的形成以及DNA链交换。DNA链成功侵入后，DNA合成利用未受损的DNA模板恢复丢失的遗传信息。在本章中，我们描述了两种成熟的生化测定方法，用于研究RecA/Rad51家族蛋白标志性的DNA链转移活性：D环测定法和DNA链交换反应。此外，我们还描述了一种将D环形成与DNA合成相结合的D环延伸测定法，可用于确定DNA聚合酶在同源重组中的作用。此外，我们还提供了一个研究蛋白质介导的DNA退火的方案，这是合成依赖性链退火（SDSA）和双Holliday连接（dHJ）途径以及单链退火（SSA）途径中的关键步骤。超螺旋质粒DNA的质量在重组同源重组反应中至关重要，本章还包括了一个描述这种DNA底物制备方法的方案。
