Improved method for the generation of double Holliday junction DNAs

Recombinational repair provides an important pathway for the repair of DNA double strand breaks that arise in mitotic and meiotic cells. Homologous pairing and strand exchange leads to the formation of DNA intermediates that are linked by double Holliday junctions, and these need to be resolved prior to chromosome segregation and cell division. In mitotic cells, resolution occurs by either of two distinct pathways (i) Nucleolytic cleavage by GEN1 or SLX1-SLX4-MUS81-EME1-XPF-ERCC1 (SMX complex), or (ii) dissolution mediated by BLM-TopoIIIα-RMI1-RMI2 (BTRR complex). To facilitate the biochemical analysis of these pathways, we previously developed a novel methodology, involving DNAzyme self-cleavage, to generate 1.8-kb long DNA molecules containing dHJs. This involved the sequential annealing of precursor ssDNAs, which were first individually isolated through multiple rounds of gel purification and ethanol precipitation, but unfortunately the method was laborious and inefficient as considerable DNA losses were incurred at each step. Here, we describe a significantly improved methodology, that increases both the efficiency and yield without impacting the quality of the dHJs produced. The new method is rapid and simple, requiring only basic molecular biology expertise, and results in the formation of dHJs that make ideal substrates for the biochemical analysis of dissolution and resolution reaction in vitro . • Recombinational repair of DNA double-strand breaks proceeds through double Holliday junction (dHJ) intermediates that must be resolved before chromosome segregation, via either nucleolytic cleavage (GEN1 or the SMX complex) or dissolution (BTRR complex). • A DNAzyme-based self-cleavage strategy was previously used to generate 1.8-kb DNA substrates containing defined dHJs for biochemical analysis. • The original protocol required multiple rounds of ssDNA purification (gel extraction and ethanol precipitation), leading to significant DNA loss and making the process labor-intensive and inefficient. • The newly described methodology substantially improves efficiency and yield while preserving the structural integrity and quality of the dHJ substrates. • The optimized approach is rapid, technically straightforward, and produces high-quality dHJs that are well suited for in vitro analysis of dissolution and resolution pathways.