Abstract
V(D)J recombination generates a remarkably diverse repertoire
of antigen receptors through the rearrangement of germline DNA.
Terminal deoxynucleotidyl transferase (TdT), a polymerase that adds
random nucleotides (N regions) to recombination junctions, is a
key enzyme contributing to this diversity. The current model is
that TdT adds N regions during V(D)J recombination by random collision
with the DNA ends, without a dependence on other cellular factors.
We previously demonstrated, however, that V(D)J junctions from Ku80-deficient
mice unexpectedly lack N regions, although the mechanism responsible for
this effect remains undefined in the mouse system. One possibility
is that junctions are formed in these mice during a stage in development
when TdT is not expressed. Alternatively, Ku80 may be required for
the expression, nuclear localization or enzymatic activity of TdT.
Here we show that V(D)J junctions isolated from Ku80-deficient fibroblasts
are devoid of N regions, as were junctions in Ku80-deficient mice.
In these cells TdT protein is abundant at the time of recombination,
localizes properly to the nucleus and is enzymatically active. Based
on these data, we propose that TdT does not add to recombination junctions
through random collision but is actively recruited to the V(D)J
recombinase complex by Ku80.
