Terminal Deoxyribonucleotidyl Transferase 97sists of 508-529 amino acids. A high degree of sequence homology
(>80%) has been observed between TdTs from different species. There
is also a remarkable similarity between TdT and the cellular repair
polymerase, DNA polymerase 13. Earlier preparations represented a
proteolytically degraded form of the enzyme that consists of two sub-
units. The smaller 8000 Da ~-subunit corresponds to amino acids
403-508 of human TdT, and the larger 24,000 Da 13-subunit corre-
sponds to amino acids 159-402 of the undegraded 60,000 Da form (3).
The undegraded form of the enzyme has been reported to exhibit a
severalfold lower turnover number than the (commercially available)
degraded enzyme (16). It is not yet known whether proteolytic degra-
dation changes enzymic properties other than kca t.
The two-subunit form of the enzyme is commercially available
from many biochemical companies. Although many firms deliver an
excellent enzyme, every now and then we have observed nuclease
contamination in different batches. Therefore, it is recommended
that the specifications of different lots be checked, particularly for
nuclease contamination, and moreover, it is wise to include experi-
mental controls that would detect exonuclease and endonuclease acti-
vity (see, e.g., [17]).
2.2. Reaction Conditions
2.2.1. Reaction Buffer
TdT is peculiar with regard to the reaction conditions in several
respects. The activity is strongly inhibited by the ammonium cation as
well as chloride, iodide, and phosphate anions stimulating a quest for
the optimal buffer. In general, potassium or sodium cacodylate buffers
are preferred, since they were shown to be optimal for polypurine and
polypyrimidine synthesis (18). However, cacodylate buffers suffer
from important drawbacks. First, cacodylate (dimethyl arsenic acid)
is toxic; second, cacodylate might be contaminated by heavy metal
ions, which must be removed prior to use, e.g., by treatment with a
complexing chelate resin, and third, SH-protective compounds, such
as DTT, which are mandatory for an enzyme's activity, react them-
selves with cacodylate to yield garlic-like smelling and highly toxic
sulfur-cacodylic compounds. Thus, an alternative for cacodylate buff-
ers is highly desirable. The only systematic study for replacing
cacodylate by other buffer substances revealed that TdT activity in