Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks

Abstract

Loss or addition of nucleotides at junctions generated by V(D)J recombination significantly expands the antigen-receptor repertoire. Addition of nontemplated (N) nucleotides is carried out by terminal deoxynucleotidyl transferase (TdT), whose only known physiological role is to create diversity at V(D)J junctions during lymphocyte development. Although purified TdT can act at free DNA ends, its ability to add nucleotides (i.e. form N regions) at coding joints appears to depend on the nonhomologous end-joining factor Ku80. Because the DNA ends generated during V(D)J rearrangements remain associated with the RAG proteins after cleavage, TdT might be targeted for N region addition through interactions with RAG proteins or with Ku80 during remodeling of the post-cleavage complex. Such regulated access would help to prevent TdT from acting at other types of broken ends and degrading the fidelity of end joining. To test this hypothesis, we measured TdT's ability to add nucleotides to endonuclease-induced chromosomal and extrachromosomal breaks. In both cases TdT added nucleotides efficiently to the cleaved DNA ends. Strikingly, the frequency of N regions at non-V(D)J-generated ends was not dependent on Ku80. Thus our results suggest that Ku80 is required to allow TdT access to RAG post-cleavage complexes, providing support for the hypothesis that Ku is involved in disassembling or remodeling the post-cleavage complex. We also found that N regions were abnormally long in the absence of Ku80, indicating that Ku80 may regulate TdT's activity at DNA ends in vivo.

Terminal deoxynucleotidyl transferase requires KU80 and XRCC4 to promote N-addition at non-V(D)J chromosomal breaks in non-lymphoid cells

“…Importantly, in contrast to a study based on an episomal plasmid assay (28), in a chromosomal context, we found no N-additions in KU-deficient cells after TdT addition (Figure 3B and C), except for one event out of 23 junctions (Figure 3B), which is likely to be TdT-independent because it was also observed in cells without TdT (Figure 3A, Table 1, P  = 0.7342). Taken together, these data show that N-addition at intrachromosomal I-SceI-DSBs is largely KU80-dependent.…”

High Frequency Targeted Mutagenesis Using Engineered Endonucleases and DNA-End Processing Enzymes

“…Tdt is a template-independent DNA polymerase that catalyzes the addition of deoxynucleotides to the 3′-hydroxyl terminus of oligonucleotide primers. It is expressed specifically in lymphoid cells during V(D)J recombination, increasing antigen receptor diversity by adding nucleotides at the coding ends of immunoglobulin and T cell receptor gene segments [32], [33], [34] Co-transfection of cells with Tdt and meganuclease leads to a 3-fold increase in GFP-positive cells (Figure 1B, compare 1.8% to 0.6% with the meganuclease alone). However, molecular analysis of the locus revealed a 8.2-±0.14 (p<0.0005) fold increase (26.9% vs. 3.2%) in the TM frequency in the Tdt co-transfected samples.…”

Terminal deoxynucleotidyl transferase: The story of a misguided DNA polymerase

“…For example, the “N” regions generated by TdT are unusually longer when Ku80 is knocked-out compared to when it is normally expressed [152]. Since Ku80 aids in the recruitment of TdT to the site of V(D)J recombination, these knock-down experiments suggests that Ku80 also regulates the catalytic activity of TdT once it is bound to DNA [61, 153].…”

“…TdT activity can also be regulated indirectly by other members of the X-family of polymerases. For example, TdT and Pol μ can efficiently compete for the same DNA substrate [152]. It has subsequently been argued that both proteins, if present during V(D)J recombination, could compete for DNA ends such that Pol μ could affect the activity of TdT during “N” region synthesis [132].…”