Preparation and Resolution of Holliday Junction DNA Recombination Intermediates

MutSβ Stimulates Holliday Junction Resolution by the SMX Complex

“…Recombination intermediates containing a single HJ (a-structures) were generated by RecA-mediated strand exchange using gapped circular pDEA-7Z and 3 0 -32 P-end labeled linearized pDEA2 plasmid DNA, as described (Shah Punatar and West, 2018). Purified a-structures were stored at 4 C in 20 mM Tris-HCl, pH 8.0, 5 mM MgCl 2 ,1 mM DTT and 100 mg/ml BSA.…”

Generation of double Holliday junction DNAs and their dissolution/resolution within a chromatin context

“…Following cleavage, the precursor ssDNA (d1–d4, s1–s4) was separated from backbone ssDNA and other DNA contaminants by gel electrophoresis and electroelution as described ( 32 ). Typically, a 180-mL 2% (wt/vol) agarose/Tris-acetate-EDTA (TAE) gel was cast in a 15 × 10 cm gel tray (Bio-Rad 1704416) with a single large sample well.…”