a, Nuclease assay was performed with a 5′ 32P-labelled 2.7-kb-long dsDNA substrate either without streptavidin (lanes 2–5) or with streptavidin (lanes 7–10), and MRX and Sae2, as indicated, for 60 min. The 2.7-kb-long substrate was prepared by reacting pATTP-S plasmid with annealed labelled oligonucleotides and ΦC31 integrase, as described in Methods. Reaction products were separated on a 15% denaturing polyacrylamide gel. Unprocessed DNA substrate did not enter the gel and remained trapped in the wells. MRX alone has the capacity to cleave dsDNA endonucleolytically at various distances from the 5′ end (lane 3), in agreement with previous reports24,33. This endonuclease activity is not affected by the protein block (compare lanes 3 and 8). Sae2 promotes endonucleolytic cleavage specifically near the protein-blocked DNA end (lane 10, indicated by red arrows). b, Assay as in a, but with a 3′-labelled DNA substrate. No endonuclease activity of MRX and Sae2 near the 3′ end was detected. c, Nuclease assay as in Fig. 3a, but with a DNA substrate of 100 bp in length (instead of 70 bp). Concerted action of MRX and Sae2 resulted in DNA cleavage ∼15–20 nucleotides away from the streptavidin-blocked 5′ DNA end. The position of the cleavage was identical for both 100- and 70-bp-long DNA substrates (compare with Fig. 3a), suggesting that the protein-blocked DNA end directs the position of cleavage by MRX and Sae2. d, A cartoon depicting the position of endonucleolytic cleavage by MRX–Sae2. For simplicity, the MRX complex is depicted as a monomer.