Figure 7.

Ku and MRX complexes oppositely regulate the nuclease activity of Exo1. (A) DNA resection assays were performed with linearized DNA as a substrate (cut with SphI) and analysed by native agarose gel electrophoresis and SYBR green staining for the double-stranded DNA (bottom panel) followed by non-denaturing Southern hybridization with a strand-specific RNA probe for the 3′ strand, as previously described (Hopkins and Paull, 2008) (top panel). Reactions contained 10 ng (0.35 nM) pNO1 DNA, 2 nM yeast wt Exo1, MRX (8.3 or 25 nM), Sae2 (8.6 or 26 nM) and 10 nM Ku heterodimer as indicated. The position of single-stranded DNA in the gel was marked as ‘ss', and that of the un-resected plasmid is marked as double stranded, ‘ds'. Migration of molecular weight markers (kb) are shown in the lane marked ‘M'. (B) A model describing how MRX and Sae2 proteins facilitate end resection at HO breaks.