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Properties of Exonucleases and Non-specific Endonucleases

This table is intended to be used as a guideline. Not all reported activities and properties for each exonuclease or endonuclease are listed. The amount of enzyme, substrate and time of incubation can have a dramatic effect upon the desired outcome of the experiment.

Enzyme Polarity Activity on ssDNA Activity on dsDNA1 Partial Digestion to generate ss Extension2 Products Produced3 Inhibition by Phosphorothioate4 Notes
linear circular linear 5' ext linear 3' ext linear blunt nicked (circular/linear) circular (supercoiled)
Exonuclease I (E. coli) 3' → 5' + — — — 15 — 5 — — No dNMP, dinucleotide 6 Yes 15, 5, 6
Thermolabile Exonuclease I 3' → 5' + — — — 15 — 5 — — No dNMP, dinucleotide 6 Yes 15, 5, 6
Exonuclease T 3' → 5' + — — — 7 — 5 — — No dNMP,dinucleotide,  short oligo Yes 5, 7
Exonuclease VII both + + 8 — 17 — 17 — — — No short oligos No 8
RecJf 5' → 3' + — — 15 — — 5 — — No dNMP, ssDNA Yes 5, 15
Mung Bean Nuclease Endonuclease + + — — — — — No dNMP, ssDNA No  
Nuclease P1 Endonuclease + + — — — — — No 5’ mononucleotides No  
Exonuclease III (E. coli) 3' → 5' +/-16 — + +/- 14 + + — 5' dNMP, ssDNA Yes 14
T7 Exonuclease 5' → 3' — — +/- + + + — 3' dNMP, dinucleotide, ssDNA 9 Yes 9
Exonuclease V (RecBCD) both + + + + + — — Yes Short oligos No  
Exonuclease VIII, truncated 5' → 3' +/- 10 — + + + — — 3' dNMP, ssDNA No 10
Lambda Exonuclease 5' → 3' +/- 10 — +/- 11 + + +/- 11 — 3' dNMP, dinucleotide, ssDNA, Yes 10, 11
Nuclease BAL-31 both 12   + + + + + + — Yes ssDNA, dNMP No 12
T5 Exonuclease 5' → 3' + + + + + + — 3' dNMP to 6 mer No  
DNase I (RNase-free) Endonuclease + + + + + + + NA dinucleotides, trinucleotides, oligonucleotides, ssDNA, dsDNA, No  
Micrococcal Nuclease Endonuclease + + + + + + + NA diphosphonucleotides, ssDNA, dsDNA 3'-monophosphonucleotides 13 No 13

Footnotes

  1. The ability to act on short extensions, blunt ends and nicks distinguishes these enzymes; some of these ends are conveniently generated by restriction digestion.  The 5' and 3' extensions tested were 4 nt in length
  2. Partial digestion of dsDNA by Lambda Exonuclease, T7 Exonuclease and Exonuclease III will produce dsDNA products with ss extensions.  Complete digestion produces ssDNA as products.
  3. Complete hydrolysis of the preferred substrate will generate the listed products
  4. To inhibit exonucleases, use of at least 5 phosphorothioate (pt) bonds in a row is recommended. These bonds must be placed at the end of the DNA corresponding to the Polarity of the enzyme; 5' end for 5'-->3' nucleases, the 3' end for 3'-->5' nucleases, and at both ends if the nucleases cannot initate at both ends. Endonucleases cannot be inhibited by pt bonds unless the entire sequence has pt bonds between all nucleotides.
  5. Depending upon the DNA sequence and amount of exonuclease, RecJf, Thermolabile Exonuclease I, Exonuclease I and Exonuclease T may remove a few nucleotides from blunt termini.
  6. Thermolabile Exonuclease I, Exonuclease I release dNMP from ssDNA, except from the last hydrolytic step where a dinucleotide is produced.
  7. Exonuclease T can be used to make 3' extensions blunt, however, the yield is low.
  8. Exonuclease VII will not be able to digest circular ssDNA when EDTA is present in the reaction. In the absence of Mg++ the enzyme will act as a pure exonuclease.
  9. It has been reported that the initial first product hydrolyzed from dsDNA by T7 Exonuclease is a dinucleotide.  Subsequent hydrolytic cleavage releases dNMP.
  10. Lambda Exonuclease and Exonuclease VIII, truncated only cut ssDNA if the 5' contains a phosphate
  11. Lambda Exonuclease has a strong preference for initiating on dsDNA containing a 5' phosphate.  Thus if linear dsDNA has a 5' phosphate at one end and lacks a 5' phosphate on the other end, then Lambda Exonuclease will preferentially degrade the DNA that contains the phosphorylated end.
  12. BAL-31 Nuclease has been reported as having both ss endonuclease activity as well as 3' to 5' exonuclease activity.  Thus any linear DNA is substrate for this enzyme.
  13. Products of Micrococcal Nuclease degradation have 3' phosphates.  Also cuts RNA whereas DNaseI does not.
  14. Exonuclease III will be inhibited by overhangs >4 nucleotides
  15. RecJf is not suitable for making 5' extensions blunt.  Thermolabile Exonuclease I and Exonuclease I are not suitable for making 3' extensions blunt.  These  enzymes require longer length ssDNA extensions to initiate than those generated by restriction enzymes.
  16. Exonuclease III exhibits 5-10x less activity on linear ssDNA versus linear dsDNA
  17. For information on removing ssDNA extensions from dsDNA see the Blunting Selection chart

 

Table Legend

+ : activity, preferred substrate
— : no significant activity
+/- : activity greatly reduced relative to preferred substrate
NA: not applicable
ss: single-stranded
ds: double-stranded
ext: extension
dNMP: deoxyribonucleoside monophosphate