Optimizing the large-scale production of Saw1 and the Saw1-Rad1-Rad10 nuclease complex for structural studies

Abstract

Yeast Rad1-Rad10 is a structure specific nuclease that processes branched double-strand break (DSB) repair intermediates; the persistence of which can impede normal DNA metabolism. The single strand annealing (SSA) mechanism of DSB repair acts when homologous repeats flank both sides of the DSB. End resection from the 5′ ends of the break exposes complementary sequences at the flanking repeats, which are annealed to form 3′ non-homologous flap structures. Saw1 recruits Rad1-Rad10 recruits to these 3′ non-homologous flaps, where Rad1-Rad10 incises the DNA and removes the flap. Saw1 has affinity towards branched DNA structures and forms a stable complex with Rad1-Rad10. The mechanism of both structure specific recruitment and nucleolytic activity of the Saw1-Rad1-Rad10 complex is currently unknown. To study this nuclease complex, we need to produce large quantities of pure, stable, and active recombinant protein. Using dynamic light scattering (DLS) and differential scanning fluorimetry (DSF)-based high throughput thermal stability assays, we have developed a method for large-scale production of recombinant Saw1. This optimized method has increased the stability and yield of protein, thereby allowing for future biochemical investigation of Saw1. Similarly, we have optimized the large-scale production of the higher molecular-weight complex (Saw1-Rad1-Rad10) and improved the homogeneity of the recombinant complex. We have also biochemically characterized the minimal branched DNA substrates for both Saw1 and Saw1-Rad1-Rad10. This work allows for biochemical investigation into the molecular mechanism of eukaryotic 3′ non-homologous flap removal during SSA.