Exonucleases are enzymes that degrade different types of DNAs in specific ways, while endonucleases cleave at specific DNA structures or modifications. Both exo- and endonucleases are useful as molecular biology tools. In this webinar, we will review the activities of exonucleases and endonucleases in more detail, provide insight on how to choose the right exo- or endonuclease for various molecular biology applications, and explain how to use these reagents when developing new molecular biology workflows.

1. Exonucleases & endonucleases
as molecular tools
Andrew Gardner, Ph.D.
Scientific Director, Molecular Enzymology Division

2. Webinar outline and goals
A. Understand exonuclease and
endonuclease activities
B. Learn how exos and endos enable
molecular biology workflows
C. Learn how to choose exos and
endos for your application

3. 1964

4. 2019
Exonucleases are
at the core of modern
molecular biology

5. Exonuclease activities
Exonucleases degrade DNA
• Polarity: 3´–>5´ or 5´–>3´
• ssDNA or dsDNA or both
• linear or nicked
• sensitive or resistant to modifications

6. Degrading ssDNA
from dsDNA
Enriching circular
from linear DNA
Creating long
ssDNA substrates
genome engineering
& hybridization
Exonucleases as molecular tools

7. Exonucleases: enriching circular DNA
Problem:
How can you separate circular DNA from linear DNA?

8. Exonucleases: enriching circular DNA
Solution:
Degrade linear DNA with
Exonuclease V (RecBCD)
Nicked circular
Closed circular
Exonuclease V

9. Exonucleases: enriching circular DNA
Example:
• Degrading contaminating chromosomal
DNA from a plasmid preparation
• Low copy plasmids
• Solution: Exonuclease V (RecBCD)
Nicked
circular
Closed
circularExonuclease V
ExoV treatment
Plasmid
Linear
chromosomal
DNA
— — + +

10. Exonucleases: enriching circular DNA
1.Nilsson, M., et al. (1994). Science 265, 2085-2088.
2.Cao, W. (2001). Clin. Appl. Immun. Rev. 2, 33-43.
3.Qi, X., et al. (2001). Nucleic Acids Res. 29, E116.
4.Cao, W. (2004). Trends in Biotechnology 22, 38-44.
5.Cheng, Y., et al. (2013). Analyst 138, 2958-2963.
6.https://genome.cshlp.org/content/23/5/843.full
Example:
• Molecular Inversion
Probes/Padlock probes
• Degrading unligated linear DNA
from circular ssDNA
• Solution: Exonuclease VII

11. PCR products
PCR primers
Problem:
How can you separate ssDNA from dsDNA?
Exonucleases: enriching dsDNA

12. Solution:
Degrade ssDNA with
Exonuclease I
Examples:
• Degradation of PCR primers in nested
PCR
- Thermolabile Exonuclease I
• Degradation of PCR primers and dNTPs
- Exonuclease I and Quick CIP
PCR products
PCR primers
Exonuclease I
Exonucleases: enriching dsDNA

13. long ssDNA substrates
• needed for CRISPR/Cas9 knock-ins
• ssDNA is less immunogenic than dsDNA
• challenge to chemically synthesize ssDNA (>200 nt)
Problem:
How can you generate long
ssDNA templates from dsDNA?
Civit, 2012
CRISPR/Cas9 knock in
ssDNA
substrate
Exonucleases: long ssDNA production

14. Solution:
Convert dsDNA to
ssDNA with Lambda
Exonuclease
Lambda exo
PCR product 1-5 kb
P
Civit, 2012
Higuchi & Ochman, 1989
CRISPR/Cas9 knock in
ssDNA
substrate
Exonucleases: long ssDNA production
P

15. Exonucleases: take homes
Exonucleases are molecular tools:
Selective degradation of DNA types:
• Degrading linear vs. circular DNA
o Plasmid from chromosomal
o Molecular Inversion Probes
• Double stranded vs. single stranded
o excess PCR primers
Converting dsDNA to ssDNA
• ssDNA substrates for hybridization
• ssDNA templates for genome engineering
12 exonucleases from New England Biolabs

16. Structure-specific
endonucleases
Modification-specific
DNA repair endonucleases
Endonucleases

17. Endonucleases: structure-specific
Flap Endonuclease I
Structure-specific
endonucleases

18. Endonucleases: modification-specific
Repair
endonucleases
DNA polymerase
nick translation
DNA ligase
DNA lesion
Base excision repair
Modification-specific
DNA repair endonucleases

19. Using DNA repair endonucleases as molecular tools

20. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
Endonucleases: gene assembly
Problem:
How can you generate specific DNA overhang
sequences for gene assembly?

21. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
Solution:
• Uracil cleavage to create unique DNA
overhang sequences
• USER Enzyme
• Thermolabile USER Enzyme II
U
U
U
U
U
USER enzyme
U
USER enzyme: UDG & Endonuclease VIII
Endonucleases: gene assembly

22. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
Example:
USER cloning for gene
fragment assembly
Endonucleases: gene assembly

23. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
Endonucleases: gene assembly
Example:
USER cloning for gene
fragment assembly

24. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
Endonucleases: gene assembly
Example:
USER cloning for gene
fragment assembly

25. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
Endonucleases: gene assembly
Example:
USER cloning for gene
fragment assembly

26. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
Endonucleases: gene assembly
Example:
USER cloning for gene
fragment assembly

27. Strand specificity is important for:
• correct annotation of genes
• identification of antisense transcripts with potential regulatory roles
• accurate determination of gene expression levels
Problem:
How to maintain strand
specificity during RNA-seq?
Endonucleases: directional RNA-seq

28. 1. Parkomchuk, D., et al. (2009)
Nucleic Acids Res. 37. e123.
2. Levin, J. Z., et al. (2010) Nature
Methods 7. 709–715.
https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
dATP, dCTP, dGTP, dUTP
Solution:
dU-strand specific cleavage
with USER enzyme
Endonucleases: directional RNA-seq

29. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
Solution:
dU-strand specific cleavage
with USER enzyme
Endonucleases: directional RNA-seq
1. Parkomchuk, D., et al. (2009)
Nucleic Acids Res. 37. e123.
2. Levin, J. Z., et al. (2010) Nature
Methods 7. 709–715.

30. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
1. Parkomchuk, D., et al. (2009)
Nucleic Acids Res. 37. e123.
2. Levin, J. Z., et al. (2010) Nature
Methods 7. 709–715.
Solution:
dU-strand specific cleavage
with USER enzyme
Endonucleases: directional RNA-seq

31. https://www.neb.com/applications/cloning-and-synthetic-biology/user-cloning/applications-of-user-and-thermolabile-user-ii-enzymes
1. Parkomchuk, D., et al. (2009)
Nucleic Acids Res. 37. e123.
2. Levin, J. Z., et al. (2010) Nature
Methods 7. 709–715.
Solution:
dU-strand specific cleavage
with USER enzyme
Endonucleases: directional RNA-seq

32. Endonucleases are molecular tools:
Structure-specific endonucleases
• Cleave at specific DNA structures
Modification-specific repair endonucleases
• DNA repair enzymes
• Utilized for sequence independent site-specific cleavage
17 endonucleases from New England Biolabs
Endonucleases: take home messages

33. Understand
biochemical activities
Exonuclease & Endonuclease R&D
Active Research and Development Program
on Exonucleases and Endonucleases
Discover
new exo and endos
Improve activities
protein engineering
Enzymes for
Innovation
www.neb.com/
EnzymesForInnovation

34. How to pick the right
exonuclease or endonuclease
for your workflow?
Choosing the right enzyme
New, intuitive and clear NEB resources to match the right enzyme with your application
goal: accelerate workflow development using endos and exos as molecular tools

35. Webpage resources
Activity illustration

36. Activity description
Webpage resources

37. Popular applications
Webpage resources

38. Exo and Endo activity illustrations
OHX5´
3´
3´
5´
5´
3´
3´
5´
ApeI
DNA
dRP
Gap
dRP= deoxyribose phosphateX = AP site
X5´
3´
3´
5´
Endonuclease III
Glycosylase
Endonuclease III
AP lyase
DNA
thymine glycol
urea
X= 5, 6-dihydroxythymine
uracil glycol
Y = AP site P = Phosphate
UA = α,β-unsaturated aldehyde
UA5´
3´
3´
5´
P
Gap
Product 2Product 1
Y5´
3´
3´
5´
X
3´
5´
Endonuclease IV
X5´
3´
DNA
Phosphate
α,β-unsaturated aldehyde (UA)
X=
X = AP site
dRP= deoxyribose phosphate
Primary activity
3´
5´
5´
3´ Secondary activity
OH5´
3´
3´
5´
dRP
Gap
dRP
OHX5´
3´
3´
5´
5´
3´
3´
5´
Endonuclease V
DNA
P
Nick
P= Phosphate
X = Major
Deoxyinosine
Minor
AP site
Urea
Mismatches
Hairpin
X
Minor flaps
Pseudo-Y
Cleaves 2nd phosphodiester
bond, 3´ to mismatch
X5´
3´
3´
5´
Endonuclease VIII
Glycosylase
Endonuclease VIII
AP lyase
DNA
Y = AP site P = phosphate
P5´
3´
3´
5´
P
Gap
Product 2Product 1
Y5´
3´
3´
5´
urea
thymine glycol
uracil glycol
methyltartronylurea
X= 5, 6-dihydroxythymine
5-hydroxy-5-methylhydanton
6-hydroxy-5,6-dihydrothymine
5´
3´
3´
5´
Thermostable FEN1
Glycosylase
DNA
OH5´
3´
3´
5´
POH
5´
5´P
OH
X5´
3´
3´
5´
5´
3´
3´
T7 Endonuclease I
DNA
– Best at C mismatches
– Doesn’t recognize all mismatches
X
X
Nick
Nick
5´
3´
3´
5´
Nick
Nick
P
OHX
OHrX5´
3´
3´
5´
5´
3´
3´
5´
RNase H2
DNA
rX = ribo A, G, C & U
P rX
X5´
3´
3´
5´
Fpg
Glycosylase
Fpg
AP lyase
DNA
Y = AP site
P5´
3´
3´
5´
P
Product 2Product 1
Y5´
3´
3´
5´
8-oxoguanineX=
UATT5´
3´
3´
5´
T4 PDG
Glycosylase
DNA
X = AP site
5´
3´
3´
5´
Product 2Product 1
5´
3´
3´
5´
TT = thymine dimer
TTPXTT
X5´
3´
3´
5´
UDG
Glycosylase
DNA
dUX= Y = AP site
Y5´
3´
3´
5´
X5´
3´
3´
5´
hAAG
Glycosylase
DNA
3mA, 7mG, dl, dXX= Y = AP site
Y5´
3´
3´
5´
Exonucleases Endonucleases

39. Exo and Endo activity selection charts
www.neb.com/tools-and-resources/selection-
charts/common-applications-for-exonucleases-
and-endonucleases
www.neb.com/tools-and-resources/selection-
charts/dna-repair-enzymes
www.neb.com/tools-and-resources/selection-
charts/properties-of-exonucleases-and-nonspecific-
endonucleases

40. Endo activity magnet

41. Exo activity poster

42. Summary
Now you have the tools to choose
the right exos and endos
Go develop new technologies!