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Rad catalyzes D loop formation
Rad51 catalyzes D-loop formation within the context of a right-handed nucleoprotein filament known as the presynaptic filament, whose assembly requires ATP binding by Rad51, although ATP hydrolysis prompts the dissociation of Rad51 protomers from the DNA ligand (Sung, 1994). D-loop formation by the presynaptic filament is enhanced by associated factors including the Swi2/Snf2 family proteins Rad54 and Rdh54 and the ssDNA-binding protein RPA (Daley et al., 2014, Heyer, 2007). Following strand invasion, de novo DNA synthesis occurs within the D-loop. Several DNA polymerases (Pols), namely, Pol δ and Pol ɛ in yeast (Deem et al., 2011, Hicks et al., 2010, Maloisel et al., 2008) and Pol η in vertebrates (Buisson et al., 2014, Kawamoto et al., 2005, McIlwraith et al., 2005), have been proposed to contribute to DNA synthesis, and among them, Pol δ has emerged as the major player (Lydeard et al., 2007, Maloisel et al., 2008, Wilson et al., 2013). Following repair DNA synthesis, the extended D-loop is resolved via one of the several mechanistically distinct pathways to yield mature recombinants of different classes (Sung and Klein, 2006, Symington et al., 2014). The involvement of Pol δ in repair DNA synthesis has been studied most extensively within the context of a HR pathway called break-induced DNA replication (BIR), in which an invading DNA strand primes DNA synthesis capable of copying an entire arm of the donor cftr channel (Fig. 1) (Costantino et al., 2014, Dilley et al., 2016, Donnianni and Symington, 2013, Lydeard et al., 2007, Saini et al., 2013, Wilson et al., 2013). Importantly, in genetic studies, Saccharomyces cerevisiae Pif1, a member of the SF1 family of helicases, has been implicated in BIR in conjunction with Pol δ (Chung et al., 2010, Saini et al., 2013, Wilson et al., 2013). Using highly purified S. cerevisiae proteins, a system that permits dissection of the mechanistic underpinnings of D-loop-primed DNA synthesis reaction has been developed (Li et al., 2009, Sebesta et al., 2011, Wilson et al., 2013). Using this system, we have shown that Pif1 greatly stimulates Pol δ-mediated DNA extension within the context of the Rad51-made D-loop. Importantly, we have furnished evidence that Pif1 fulfills two distinct functions in the DNA synthesis reaction; namely, (i) it enhances the ability of the polymerase ensemble to catalyze DNA strand displacement synthesis via an interaction with the proliferating cell nuclear antigen (PCNA), the polymerase processivity clamp, and (ii) concomitant with DNA synthesis, Pif1 dissociates the invading strand to establish a migrating DNA bubble structure (Fig. 1) (Wilson et al., 2013). This chapter describes the materials and experimental procedures for reconstituting the repair DNA synthesis reaction using a ssDNA oligonucleotide as the invading strand and supercoiled dsDNA as the information donor. Our method utilizes Rad51, RPA, and Rad54 to generate D-loops and PCNA, the multisubunit PCNA loader replication factor C (RFC), the trimeric Pol δ and Pif1 helicase in the DNA synthesis phase of the reaction. The methods for product analyses are also described.