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 |
| 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
- 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
- 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.
- Complete hydrolysis of the preferred substrate will generate the listed products
- 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.
- 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.
- Thermolabile Exonuclease I, Exonuclease I release dNMP from ssDNA, except from the last hydrolytic step where a dinucleotide is produced.
- Exonuclease T can be used to make 3' extensions blunt, however, the yield is low.
- 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.
- It has been reported that the initial first product hydrolyzed from dsDNA by T7 Exonuclease is a dinucleotide. Subsequent hydrolytic cleavage releases dNMP.
- Lambda Exonuclease and Exonuclease VIII, truncated only cut ssDNA if the 5' contains a phosphate
- 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.
- 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.
- Products of Micrococcal Nuclease degradation have 3' phosphates. Also cuts RNA whereas DNaseI does not.
- Exonuclease III will be inhibited by overhangs >4 nucleotides
- 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.
- Exonuclease III exhibits 5-10x less activity on linear ssDNA versus linear dsDNA
- 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
