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|
|Msz 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|
|DNase I-XT||Endonuclease||+||+||+||+||+||+||+||NA||dinucleotides, trinucleotides, oligonucleotides, ssDNA, dsDNA,
|Micrococcal Nuclease||Endonuclease||+||+||+||+||+||+||+||NA||diphosphonucleotides, ssDNA, dsDNA 3′-monophosphonucleotides 13||No||13|
- 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 initiate 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, Msz Exonuclease I, and Exonuclease T may remove a few nucleotides from blunt termini.
- Thermolabile Exonuclease I, Exonuclease I, and Msz 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 DNase I does not.
- Exonuclease III will be inhibited by overhangs >4 nucleotides
- RecJf is not suitable for making 5′ extensions blunt. Thermolabile Exonuclease I, Exonuclease I, and Msz 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
dNMP: deoxyribonucleoside monophosphate