A sequence-dependent exonuclease activity from Tetrahymena thermophila

Abstract

Background: Telomere function requires a highly conserved G rich 3'- overhang. This structure is formed by 5'-resection of the C-rich telomere strand. However, while many nucleases have been suggested to play a role in processing, it is not yet clear which nucleases carry out this 5'-resection.

Results: We used biochemical purification to identify a sequence-dependent exonuclease activity in Tetrahymena thermophila cell extracts. The nuclease activity showed specificity for 5'-ends containing AA or AC sequences, unlike Exo1, which showed sequence-independent cleavage. The Tetrahymena nuclease was active on both phosphorylated and unphosphorylated substrates whereas Exo1 requires a 5'-phosphate for cleavage.

Conclusions: The specificities of the enzyme indicate that this novel Tetrahymena exonuclease is distinct from Exo1 and has properties required for 3'-overhang formations at telomeres.

Figure 1

Design of oligonucleotide substrates and detection of a telomere-processing nuclease activity. (A) Schematic representation of the substrate structures utilized in detecting the telomere-processing nuclease in Tetrahymena extracts. Solid lines represent non-telomeric sequences, and the numbers indicate lengths of non-telomeric base pairs in the duplex region. Each gray circle represents a TTGGGG repeat unit, and each black circle is a complementary CCCCAA repeat unit. S inside a circle represents the streptavidin protein. The stars indicate the approximate location of radiolabels, and 4-prong symbols are biotinylation sites. The annealing components were: Ia-S1*:S18; Ib-S1:S18*; IIa-S2*:S18; IIb-S2:S18*; IIIa-S1*:S19; IIIb-S1:S19*; IVa-S3*:S20; IVb-S3:S20*; Va-S3*:S21; Vb-S3:S21*; VIa-S4*:S20; VIb-S4:S20*. (B) Tetrahymena crude cell extracts were assayed with Ib and IVb and with/without streptavidin. ~ 1.2 μg total proteins from the extracts were incubated in each reaction for 15 min. (C) Nuclease purification scheme is presented. (D) The nuclease is 100 kDa in size. Chromatographs of gel filtration runs for standards and nuclease samples are shown in a superimposed graph. The peaks of standard proteins are labeled with numbers: peak 1-bovine thyroglobulin, 670,000; peak 2-bovine γ-globulin, 158,000; peak 3-chicken ovalbumin, 44,000; peak 4-horse myoglobin, 17,000; peak 5-vitamin B12, 1,350. The arrow indicates where the nuclease activity was eluted off the column. The apparent molecular weight was calculated as described under Methods. The activity corresponding to the fractions were indicated in the second panel with ~ 300-600 ngs protein per reaction.

Figure 2

Enzyme titration and time course of purified telomere-specific nuclease that is a DNase. The triangles indicate increasing enzyme concentrations or time. (A) An enzyme titration of the purified telomeric nuclease (0, 0.95, 1.9, 4.75, 9.5, and 15.8 ng) is shown with substrates Ib and IVb. The reactions were incubated for 20 min. (B) Time course (0, 5, 10, 15, 20, and 30 min.) of purified telomeric nuclease at 4.75 ng per reaction with substrates Ia, Ib, IVa, and IVb is shown. (C) Cleavage of DNA but not RNA. Boxed sequences are non-telomeric ribonucleotides. Open gray circles are GGGGUU ribonucleotide repeat units, and open black circles are AACCCC ribonucleotide repeat units. Substrates (left to right) are Ib, M1:S18*, IVb, M2:S20*, S1:M3*, M1:M3*, S3:M4*, and M2:M4*.

Figure 3

Tetrahymena nuclease is an exonuclease. (A) Enzyme titrations (0, 0.46, and 2.3 ng) were incubated for 20 min. (on a 15% urea gel). Substrates (left to right) are Ib, IVb, S16:S33*, and S17:S34*. (B) Enzyme titration (0, 0.95, and 9.5 ng) of enriched nuclease with end-labeled non-telomeric, S1:S18 (lanes 1-3), and telomeric, S3:S20 (lanes 4-6), substrates (on a 15% urea gel). Arrow indicates an unique cleavage product of this nuclease. (C) Internally-labeled phosphorothioate substrates (with 0.9 ng of enzyme and substrates Ib, IVb, and various blockage locations on IVb, S3:S20) and (D) end-labeled phosphorothioate substrates (with 20 ng of enzyme and substrates S1:S18, S3:S20, and various single-bond blockage on S3:S20) are shown. Arrow indicates an unique cleavage product of this nuclease that is blocked by terminal phosphorothioate. Both assays were resolved on 15% urea gels.

Figure 4

Cleavage and substrate specificity of telomere-specific nuclease activity. An enzyme titration (represented by triangles) using, 0, 0.46, and 2.3 ng (in A) or 0, 0.23, 0.93 ng (in B) was performed for a 20-min. incubation time. (A) Substrates (left to right) are with various repeat permutations: Ib, IVb, S9:S26*, S10:S27*, S11:S28*, S12:S29*, and S13:S30* (on a 15% urea gel). (B) Substrates (left to right) are Ib, IVb-Tetrahymena GGGGTT repeat, S14:S31*-mammal AGGGTT repeat, S15:S32*-yeast G_1-3T repeat, and S8:S25*-GACGAT random repeat (on a 15% urea gel).

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*.

Figure 6

Enriched Tetrahymena nuclease shows sequence-dependant cleavage. Activity of Tetrahymena nuclease (30 ng) and Exo1 (1 fmol or 0.04 ng) with various unique sequences that end in all combinations of two terminal nucleotides, either unphosphorylated (A) or phosphorylated (B) were tested. Reactions were performed for 20 min and resolved on 10% urea denaturing polyacrylamide gels. All assays were run and performed on the same day, and the time for exposure to phosphorimager screens was the same. Substrates used are: S1:S18* (lanes 1-3), S3:S20* (lanes 4-6), S35:S57* (lanes 7-9), S36:S58* (lanes 10-12), S37:S59* (lanes 13-15), S38:S60* (lanes 16-18), S39:S61* (lanes 19-21), S40:S62* (lanes 22-24), S41:S63* (lanes 25-27), S42:S64* (lanes 28-30), S43:S65* (lanes 31-33), S44:S66* (lane 34-36), S45:S67* (lanes 37-39), S46:S68* (lanes 40-42), S47:S69* (lanes 43-45), S48:S70* (lanes 46-48), S49:S71* (lanes 49-51), S50:S72* (lanes 52-54), S51:S73* (lanes 55-57), S52:S74* (lanes 58-60), S53:S75* (lanes 61-63).