Ligases 223
- 10X Ligation buffer:
- 200 mM Tris-HC1, pH 7.6
- 50 mMMgC12
- 50 mM DTT
- 500 pg/mL BSA.
- 10 mM ATP
3.6.2. Protocol - Prepare the fragments to be ligated by digestion of the source DNA
species (e.g., vector and foreign genomic DNA) with restriction endo-
nucleases. Purify by phenol/chloroform extraction and ethanol precipi-
tation. Resuspend each DNA in 10 mM Tris/1 mM EDTA, pH 7.6. - Mix aliquots containing 0.1 ~g of each DNA species. Add 15 laL water.
Warm to 70°C for 10 min, to melt any annealing of cohesive ends
between molecules of the same species. Allow to cool slowly to room
temperature, and then place on ice. - Add 2 lxl-, of 10X ligation buffer, plus 1 IxL 10 mM ATP.
- Add 0.1 Weiss unit T4 DNA ligase.
- Incubate at 16°C for 4 h.
- Terminate the reaction by heating to 70°C for 10 min. Aliquots of the
ligation mixture can then be used for bacterial transformation either
directly or after ethanol precipitation.
4. T4 RNA Ligase (EC 6.5.1.3)
Like T4 DNA ligase, this enzyme is found in E. coli after infection
with T-even phage (57). It has also been cloned and is now available
from several overproducing strains of E. coli. The enzyme forms a
phosphodiester bond between a phosphorylated 5' terminus and a 3'
hydroxyl similarly to the DNA ligases (58-60), except that the sub-
strates in this case are in general RNA moieties.
The in vivo role of the enzyme is unclear, although it is probably
involved in host RNA modification and transcript splicing (61). The
protein does have a morphological role in the noncovalent attachment
of tail fibers to the base plate of the phage (62).
4.1. The Enzyme
The mol mass of the enzyme has been determined as 48,000 Da by
sedimentation equilibrium, 47,000 Da by gel filtration, and 41,000-
45,000 Daby SDS-PAGE (63,64). It is monomeric in solution (63) and
has an isoelectric point of 6.1 (65).