Part:BBa_K676006

T5 Phage Exonuclease

The bacteriophage T5 exonuclease is a unique DNase which is ideal for plasmid DNA manufacturing. This nuclease can digest linear single and double stranded DNA, but will leave supercoiled plasmids alone. Furthermore, this DNase can also digest irreversibly denatured plasmids which resemble supercoiled plasmids and are otherwise resistant to digestion by restriction enzymes. The D15 exonuclease has a 5’ to 3’ activity, which is complementary to E. coli exonuclease III with a 3’ to 5’ activity [1]. Also compared to other similar DNases like, E. coli exonuclease III and T7 phage exonuclease, which can only digest double-stranded DNA, D15 can digest both single and duplex DNA molecules. Experiments carried out by Sayers et al. have substantially proved the absence of double-stranded endonuclease activity of the D15 nuclease and which is why this exonuclease does not affect closed circular supercoiled plasmids at all and is therefore referred to as a ‘plasmid-safe nuclease’. Interestingly, this particular exonuclease shares 54% similarity to the E. Coli DNA polymerase I. So it can be concluded that the T5 exonuclease is a very useful tool in plasmid DNA manufacturing, as it reduces the complexity and length of the entire downstream process, by getting rid of ‘junk plasmids’ (nicked and linear forms), which have less therapeutic value than supercoiled plasmids [2].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 600
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Sayers, J.R. and F. Eckstein, Properties of Overexpressed Phage-T5 D15 Exonuclease - Similarities with Escherichia-Coli DNA-Polymerase-I 5'-3' Exonuclease. Journal of Biological Chemistry, 1990. 265(30): p. 18311-18317.

2. Sayers, J.R., D. Evans, and J.B. Thomson, Identification and eradication of a denatured DNA isolated during alkaline lysis-based plasmid purification procedures. Analytical Biochemistry, 1996. 241(2): p. 186-189.