Defining the mechanistic basis of ATM’s impact on VDJ recombination

项目摘要 ATM激酶是DNA损伤反应的核心;因此，这种大激酶是调节DNA损伤的核心。 在VDJ重组过程中，发育中的淋巴细胞如何对自身施加的DNA损伤作出反应。虽然 证据是压倒性的和明确的，ATM有助于精确的非同源末端连接， VDJ编码片段，并抑制其参与基因组易位，这是一个明确的机制， 对ATM实际上是如何完成这项工作的了解还很缺乏。 最近，我们发现在许多培养的细胞株中ATM消融导致附加体中VDJ连接增加， 如果ATM在末端连接中具有直接的功能作用，则这是完全违反直觉的结果。我们 认为如果ATM的作用是调节RAG后切割复合物，ATM的丢失可能 导致VDJ重组中间体的释放增加和更快的连接，解释了VDJ重组中间体的增加。 观察到的重组。我们微调了检测方法，使结构/功能，还原主义的方法可以 用于描述ATM是否以及如何直接影响RAG复合体。我们发现ATM信号抑制 连接需要RAG 1和RAG 2的非核心C-末端。这促使人们对这些地区进行了研究 潜在的ATM/DNA-PK靶位点。屏蔽这些站点不仅可以屏蔽ATM对信号接入的影响， 细胞试验中，它也消除了RAG 1/RAG 2在高度纯化和全功能RAG上的稳健磷酸化 通过ATM和DNA-PK在体外的复合物。这些数据支持我们的模型，ATM直接调节VDJ 通过RAG复合物的磷酸化进行重组。本申请中提出的实验将使用 生物化学方法来定义ATM如何影响切割和信号末端释放，并将使用细胞 染色体VDJ重组模型，以进一步确定ATM如何调节VDJ重组。

Project Summary The ATM kinase is central to the DNA damage response; it follows that this large kinase is central to regulating how developing lymphocytes respond to their self-imposed DNA damage during VDJ recombination. Although the evidence is overwhelming and unequivocal that ATM contributes to accurate non-homologous end joining of VDJ coding segments, and restrains their participation in genomic translocations, a clear mechanistic understanding of how ATM actually does this job is lacking. Recently, we found that ATM ablation in many cultured cell strains results in increased VDJ joining in episomal assays, a completely counter-intuitive result if ATM were to have a direct functional role in end joining. We considered that if ATM's role was instead, to regulate the RAG post cleavage complex(s), loss of ATM might result in increased release of VDJ recombination intermediates and more rapid joining, explaining the increased recombination observed. We fine-tuned the assay so that a structure/function, reductionist approach could be employed to delineate if and how ATM directly affects the RAG complex. We find that ATM inhibition of signal joining requires the non-core C-termini of both RAG1 and RAG2. This prompted an examination of these regions for potential ATM/DNA-PK target sites. Blocking these sites not only blocks ATM's effect on signal joining in cellular assays, it also ablates robust phosphorylation of RAG1/RAG2 on highly purified and fully functional RAG complexes by ATM and DNA-PK in vitro. These data support our model that ATM directly regulates VDJ recombination by phosphorylation of the RAG complex. The experiments proposed in this application will use biochemical approaches to define how ATM affects cleavage and signal end release, and will use a cellular model of chromosomal VDJ recombination to further define how ATM regulates VDJ recombination.