New England Biolabs: Reagents for the Life Sciences Industry

Properties of Exonucleases and 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.

EnzymePolarity³DNA substrateActivity without 5' phosphateAble to Initiate on DNA with¹Partial Digestion of ss Extension²Products Produced³End Labeled FAM cleavage⁴Phosphorothioate cleavage5Units for 90 percent digestion of 2 oligos6
ssds5' ext3' extbluntnick
Lambda Exonuclease5' → 3'+/-++/-⁷+/-YesYesNo3'dNMP and ssDNA+-2
RecJf5' → 3'+-Yes+/- ⁹No+/- ¹⁰NoNAdNMP+/-->1500
Exonuclease III (E. coli)3' → 5'+/-+YesYes+/-YesYes5'ssDNA and dNMP+-10
Exonuclease I (E. coli)3' → 5'+-YesNo+/- ⁹+/- ¹⁰NRNAdNMP and dinucleotide¹¹+-20
Exonuclease T3' → 5'+-YesNoYes¹²+/- ¹⁰NRNAdNMP+->100
Exonuclease V (RecBCD)both++YesYesYesYesNoYesshort oligos+NANA
Exonuclease VIII, truncated5' → 3'+/-+YesYesYesYesNo3'dNMP and ssDNANA+NA
Exonuclease VIIboth+-++/-+/-NoNo5'short oligosNA+10
Nuclease BAL-313' → 5' and Endonuclease¹³++YesYesYesYesYesNAssDNA and dNMPNANANR
Mung Bean NucleaseEndonuclease+-YesYesYes--NAdsDNA, dNMP and ssDNANANA10
DNase I (RNase-free)Endonuclease++NANANANANANAdsDNA, ssDNA, oligonucleotides, dinucleotides and trinucleotidesNANA0.1
Micrococcal NucleaseEndonuclease++NANANANANANAssDNA, dsDNA 3', monophosphonucleotides and diphosphonucleotides¹⁴NANANR
T5 Exonuclease5' → 3'++YesYesYesYesYesNAdNMP to 6 mer+NA2
T7 Exonuclease5' → 3'+/-+Yes+/-YesYesYes3'ssDNA, dNMP and dinucleotide⁸+-10
T7 Endonuclease IEndonuclease-+NANANANA+/-NAdsDNANANANA
Thermostable FEN1EndonucleaseNANAYesNoNoNoNoNANANANA

Table Legend

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


  1. The ability to act on short extensions and blunt ends distinguishes these enzymes; such 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 a product. 
  3. Complete hydrolysis of the preferred substrate will generate the listed products. 
  4. The ability of an exonuclease to initiate on the end of the preferred DNA substrate (ss or ds) containing a fluorescein group linked to either the 5´ or 3´ end. Phosphoramidite chemistry was used to synthesize oligonucleotides with FAM groups. The 5´ FAM was added to the oligonucleotide as a [(3´,6´-dipivaloylfluoresceinyl)-6-carboxamidohexyl]-l-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite while the 3´ FAM oligonucleotides were synthesized using the 1-dimethoxytrityloxy-3-[O-(N-carboxy-(di-O-pivaloyl-fluorescein)-3-aminopropyl)]-propyl-2-O-succinoyl-long chain alkylamino-CPG support. 
  5. The Sp stereoisomer of the phosphorothioate linkage is known to greatly inhibit cleavage of many nucleases while the Rp stereoisomer shows either no or less inhibition. Synthetic oligonucleotides containing approximately an equal ratio of both isomers necessitates the use of multiple phosphorothioates to block cleavage. The presence of 6 consecutive phosphorothioates on oligonucleotides of the preferred substrate blocked all exonucleases effectively (5% or less degradation) except T7 exonuclease which had approximately 10% cleavage. Analysis of exonucleases with oligonucleotides having either one or two consecutive phosphorothioates revealed significant degradation (not shown). 
  6. The amount of enzyme in units to cleave greater than 90% of 2 µM 35-mer oligonucleotide (s) of the preferred substrate (ss or dsDNA) in a 10 µl volume using the unit digest reaction conditions. All enzymes tested except for Exo T and RecJf could effectively cleave the substrate to completion. 
  7. Lambda exonuclease has a strong preference for initiating degradation 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 from the phosphorylated end. 
  8. It has been reported that the initial first product hydrolyzed from dsDNA by T7 Exonuclease is a dinucleotide. Subsequent hydrolytic cleavage releases dNMP. 
  9. RecJf is not suitable for making 5´ extensions blunt. Exo I is not suitable for making 3´ extensions blunt. Both RecJf and Exo I require longer length ssDNA extensions to initiate than those generated by restriction enzymes. 
  10. Depending upon the DNA sequence and amount of exonuclease, RecJf, Exo I and Exo T may remove a few nucleotides from flush termini.
  11. Exo I releases dNMP from ssDNA, except at the last hydrolytic step where a dinucleotide is produced. 
  12. Exo T can be used to make 3´ extensions blunt: however, it yields 2–4 fold fewer ligatable blunt ends when compared to Klenow polymerase plus dNTP on a test substrate. 
  13. BAL31 has been reported as having both ss endonuclease activity as well as 3´→5´ ds exonuclease activity. Thus, any linear DNA is a substrate for BAL31. 
  14. Products of Micrococcal Nuclease degradation have 3´ phosphates. 
  15. T7 endonuclease recognizes and cleaves non-perfectly matched DNA, cruciform DNA, Holliday structures or junctions. It will act more slowly on nicked dsDNA.