Published online 26 March 2004
Nucleic Acids Research, 2004, Vol. 32, No. 6 1866-1873
© 2004 Oxford University Press
Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks
Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA, 1 Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA and 2 Skirball Institute of Biomolecular Medicine and Department of Pathology, New York University School of Medicine, New York, NY 10016, USA
*To whom correspondence should be addressed. Tel: +1 713 798 5760; Fax +1 713 796 9438; Email: jwilson{at}bcm.tmc.edu Correspondence may also be addressed to David B. Roth. Tel: +1 212 263 0945; Fax: +1 212 937 2433; Email: roth{at}saturn.med.nyu.edu
Received January 9, 2004; Revised and Accepted March 3, 2004
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.
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