Nuclease S1, isolated from certain Neurospora and Aspergillus species, specifically hydrolyzes both terminal and internal phosphodiester bonds of single-stranded DNA and RNA. It is used to eliminate non-annealed polynucleotide tails and hair-pin loops in DNA-RNA or DNA-DNA duplexes in hybridization studies and in genetic recombination experiments.
Stability/Storage: For long term storage in solution, for up to six months, dilute NUCSI to ≥6000 u/ml in water and freeze in aliquots. Dilute solutions can be stabilized by adding 0.1% albumin and 10% glycerol.
Characteristics of Nuclease, S1
Molecular Weight: Approximately 32,000 - 36,000 daltons, exists as a monomer.
Optimal pH: 4.0 - 4.6
Activators: Zn2+ and/or Ca2+.
Inhibitors: EDTA, citrate and a high concentration of SDS.
Nuclease, S1 Assay
Method
One unit is the amount of enzyme liberating 1µg (0.033 A260) of acid-soluble nucleotides from heat-denatured DNA per minute at 37ÁC and at pH 4.6
Reagents
Buffer: 0.2M NaCl, 0.002M ZnCl2, 0.06M CH3COONa, pH 4.6: dissolve 5.844 gms NaCl (MW 58.44), 136mg ZnCl2 (MW 136.29) and 1.85ml concentrated glacial acetic acid in 450ml reagent grade water. Adjust pH to 4.6 with 10M NaOH. Bring to a final volume of 500ml with reagent grade water.
Enzyme Diluent: Dissolve 40mg BSA in 200 ml Buffer
Substrate: Shred into small fibers, 60mg calf thymus DNA and dissolve in 50ml reagent grade water by standing at room temperature for at least 18 hours. Additional stirring may be necessary to effect soluiton. Remove 10ml DNA solution to 10ml of buffer. This is native calf thymus DNA solution (Substrate B)
Heat the remaining DNA solution in a large Pyrex test tube with a stir bar in boiling water on a heater/stirrer while stirring for 20 minutes. Immediately pour into PRE-FROZEN 1 liter beaker on ice. Mix equal volumes of the DNA solution and cold buffer. This is heat-denatured calf thymus DNA solution (Substrate A). Use as soon as possible to prevent the blank from elevating.
15% Perchloric Acid: Add 21.5ml concentrated perchloric acid (70%) to 78.5ml deionized water.
Procedure
1. To clean glass tubes (two for each point) add 2ml Substrate B for tests. Include 2 tubes with 2ml substrate B for blanks (no enzyme added) and 2 tubes with 2ml Substrate A (native DNA test).
2. Incubate for 5 minutes before adding the enzyme.
3. Add 0.1ml enzyme dilution
4. Incubate at 37°C for 10 minutes.
5. Stop reaction by adding 2ml 15% perchloric acid.
6. Leave on ice for 10 minutes.
7. Centrifuge on a bench-top centrifuge for 15 minutes at 2000 rpm.
8. Withdraw 3ml supernatant and read A260.
Calculation
Where 1242 is a factor derived by dividing the reaction volume (4.1 ml) by the A260 of 1 µg (0.033) and dividing by the enzyme sample volume used (0.1 ml).
Biochem/physiol Actions：
Hydrolyzes only single-stranded DNA and RNA without apparent specificity for bases. Catalytic activity: Endonucleolytic cleavage to 5'-phosphomononucleotide and 5'-phosphooligonucleotide end-products. Cofactor: Zn2+. Note: Binds 3 divalent metal cations. Activity regulation: Inhibited by inorganic phosphate (Pi).
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