Exonucleases

Cuts from an end, not in the middle, so an intact circle is immune.

Four questions define an exonuclease

More-useful Exonucleases

New England Biolabs table of exonuclease properties: polarity, activity on single- and double-stranded DNA, and the products released
Adapted from the NEB properties-of-exonucleases table
Will T5 Exonuclease cut?
→ Yes. From both ends. T5 initiates on blunt ends and on 3’ extensions
Enzyme Polarity Activity on ssDNA Activity on dsDNA
linearcircular linear 5' extlinear 3' extlinear blunt nicked (circular/linear)circular (supercoiled)
T5 Exonuclease 5' → 3'++ ++++,
5’-gatttctgGAATTTGACACGTG-3’
3’-ctaaagacCTTAA-5’
Wording and values retyped verbatim from the NEB exonuclease selection chart, neb.com

T5 Exonuclease in Gibson Assembly

Lambda Exonuclease wants a 5’ phosphate

Exonuclease I: why ExoSAP exists

Enzyme Polarity Activity on ssDNA Activity on dsDNA
linearcircular linear 5' extlinear 3' extlinear blunt nicked (circular/linear)circular (supercoiled)
Exonuclease I (E. coli) 3' → 5'+, , , , , ,
Wording and values retyped verbatim from the NEB exonuclease selection chart, neb.com

Blunting a sticky end

5’-cttcgGGATCCgctggcaga…-3’
3’-gaagcCCTAGGcgaccgtct…-5’
↓ BamHI
5’-cttcgG              GATCCgctggcaga…-3’
3’-gaagcCCTAG    +         Gcgaccgtct…-5’
↓ Mung Bean Nuclease, single-stranded DNA only
5’-cttcgG                  Cgctggcaga…-3’
3’-gaagcC        +         Gcgaccgtct…-5’
↓ T4 DNA Ligase; any blunt end joins any other
5’-cttcgGCgctggcaga…-3’
3’-gaagcCGcgaccgtct…-5’

The BamHI site is gone, and four base pairs with it