Figure 4.

5′-flap cleavage by Slx1–4. (A) The indicated Y and 5′-flap substrates were incubated with Slx1–4, and the reaction products were resolved by sequencing gel electrophoresis (lanes 3,4). Comparison to a chemical sequencing ladder of 5′-[³²P]-labeled oligonucleotide 891 (lanes 1,2) revealed that hydrolysis occurred mainly at a single site (5′-GAGC↓CCTA-3′) on both substrates. Substrates were assembled as follows: Y, 888/*891; 5′ flap, 888/*891/992. R, G + A; Y, T + C. (B) Schematic diagrams indicate the sites of cleavage on three 5′-flap substrates of varying sequence adjacent to the branch site. (C) The indicated 5′-flap substrate (888/891/*992) was prepared in which the 24-nt downstream oligonucleotide was ³²P-end-labeled (lane 1). The substrate was either incubated with Slx1–4 (lane 2) or incubated with Slx1–4 followed by DNA ligase (lane 3). Products were analyzed by sequencing gel electrophoresis.