Figure 2.
Licensing of 5′ strand cleavage at a nucleosome and other internally bound obstacles by Sae2. (A) The 232-bp dsDNA (1 nM) was incubated with 40 nM MRX, 480 nM Sae2, and 64 nM Ku. (B) Reactions were carried out as in A except with the 232-bp dsDNA containing a site-specific nucleosome. The nucleosomal substrate was generated using a 2:1 molar ratio of histone octamer to DNA. (C) The two 95-bp substrates with 5-nt 3′ overhangs (2 nM each) containing an EcoRI site at either the DNA end or an internal site were tested with 24 nM EcoRI-E111Q, 40 nM MRX, and 480 nM Sae2. The asterisk denotes the 32P label in the substrate in all of the figure parts. See also Supplemental Figure S3.