Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks.
Distinct requirements for Ku in N nucleotide addition at V(D)J- and non-V(D)J-generated double-strand breaks.
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在 V(D)J 和非 V(D)J 生成的双链断裂处添加 N 核苷酸时对 Ku 的要求不同。
DOI：
10.1093/nar/gkh502
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发表时间：
2004
影响因子：
14.9
通讯作者：
Wilson,JohnH
中科院分区：
文献类型：
--
作者：
Sandor,Zoltan;Calicchio,MonicaL;Sargent,RGeoffrey;Roth,DavidB;Wilson,JohnH
通过V（D）J重组产生的连接处的核苷酸的缺失或添加显著地扩展了抗原受体库。通过末端脱氧核苷酸转移酶（TdT）添加非模板（N）核苷酸，其唯一已知的生理作用是在淋巴细胞发育期间在V（D）J连接处产生多样性。虽然纯化的TdT可以作用于游离DNA末端，但其在编码接头处添加核苷酸（即形成N区）的能力似乎取决于非同源末端连接因子Ku 80。由于V（D）J重排过程中产生的DNA末端在切割后仍与RAG蛋白结合，因此TdT可能在切割后复合物重塑过程中通过与RAG蛋白或Ku 80相互作用而被靶向用于N区添加。这种受管制的接入将有助于防止TdT作用于其他类型的断头并降低末端连接的保真度。为了验证这一假设，我们测量了TdT将核苷酸添加到核酸内切酶诱导的染色体和染色体外断裂的能力。在这两种情况下，TdT有效地将核苷酸添加到切割的DNA末端。值得注意的是，非V（D）J-生成末端的N区域的频率不依赖于Ku 80。因此，我们的研究结果表明，Ku 80是允许TdT进入RAG切割后复合物所必需的，为Ku参与分解或重塑切割后复合物的假设提供了支持。我们还发现，在没有Ku 80的情况下，N区异常长，表明Ku 80可能在体内调节DNA末端TdT的活性。
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 endsin vivo.