Figure 5

The Tetrahymena nuclease shows sequence specificity and higher activity with substrates containing 5'- phosphates. (A) Activity of enriched Tetrahymena nuclease (30 ng) on non-telomeric (Ib) and telomeric (IVb) substrates with and without 5'-phosphate is shown (lanes 5-8 and lanes 1-4, respectively) (20 min reactions). (B) Enzyme titration (0, 0.01, 0.1, 0.5, 1, 5, 10, 50, 100 fmol) of Exo1 purified protein with phosphorylated internally-labeled substrates S1:S18* (lanes 1-9) and S3:S20* (lanes 10-18) (20 min reactions on a 15% urea gel). (C) Activity of Tetrahymena nuclease (30 ng) and Exo1 (1 fmol or 0.04 ng) was assayed. Unique sequence (lanes 1-9), telomeric repeats (lanes 10-18), and AA-ending unique sequence (lanes 19-27) in either blunt, 3'-overhang, or 5'-overhang structures (10 min reactions on a 10% urea gel) are tested. Substrates (left to right) are: Ib, S54:S18*, S55:S18*, IVb, S34:S20*, S33:S20*, S52:S74*, S56:S74*, and S55:S74*.