END JOINING REACTION IN DNA REPAIR & V(D)J RECOMBINATION

描述(改编自调查人员摘要)：如果DNA双链 断裂(DSB)修复不当，可能导致染色体 易位和癌症。DSB是由外源因子产生的，例如 电离辐射(IR)，或通过V(D)J的内生过程 重组，重组免疫球蛋白和T细胞受体基因以 产生免疫多样性。值得注意的是，哺乳动物细胞利用 DSB修复和V(D)J重组的末端连接反应相同。这个 末端连接反应必须包括两个DNA末端的突触步骤， 通过微同源碱基配对对齐的末端的处理，以及 连接成共价连接的DNA双链。这一切所需的蛋白质 该实验室和其他实验室已经确定了这一过程，包括 DNA依赖蛋白激酶(DNA-PK)和XRCC4。DNA-PK由一个 与DNA末端结合的调节亚基Ku和催化亚基， 被DNA激活的p460结束。XRCC4可能通过以下途径在途径后期起作用 刺激最终的结扎反应。已经制定了一个模型，用于 Ku和p460是如何参与末端连接的。要测试该模型，特定的 这项提案的目的是调查： 1.Ku和p460在DNA末端突触中的作用一辆超大的 检测到DNA、Ku和p460的络合物，并将其表征为 确定它是否代表两个DNA分子的突触。A 19埃 获得了p460的分辨结构，提示突触可能发生在 平行的方向。实验将区分平行和非平行的 反平行突触。 2.Ku和p460在DNA末端加工中的作用。DNA-PK Ku的磷酸化激活了它的ATPase活性，这将被测试 在处理DNA末端过程中的可能作用：链交换、末端解旋、 解开连接到微同源碱基配对，并打开发夹末端。 突变的Ku的ATP结合基序将被导入Ku突变体 以测试细胞中ATPase在体内的作用。 3.末端连接反应的后期步骤。Ku与P460的络合物 DNA末端被发现可以招募额外的因子。这些因素将 通过蛋白质纯化和测试特定的候选者来鉴定， 包括XRCC4、DNA连接酶I和IV、RPA、HMG1和HMG2。曾经的首字母 已经确定了步骤的特征，将对完整的 体外末端连接反应。最终，这些研究将导致 DSB修复和V(D)J重组的分子描述，导致 对癌症和人类免疫系统有更深入的了解。

DESCRIPTION (Adapted from Investigator's abstract): If DNA double-strand breaks (DSBs) are repaired improperly, they may lead to chromosome translocations and cancer. DSBs are created by exogenous agents such as ionizing radiation (IR), or by the endogenous process of V(D)J recombination, which rearranges immunoglobulin and T cell receptor genes to generate immunological diversity. Remarkably, mammalian cells utilize the same end-joining reaction for both DSB repair and V(D)J recombination. The end-joining reaction must include steps for synapsis of two DNA ends, processing of the ends for alignment by microhomology base pairing, and ligation into a covalently linked DNA duplex. Proteins required for this process have been identified by this and other laboratories and include DNA-dependent protein kinase (DNA-PK) and XRCC4. DNA-PK consists of a regulatory subunit, Ku, which binds to DNA ends, and a catalytic subunit, p460, which is activated by DNA ends. XRCC4 may act late in the pathway by stimulating the final ligation reaction. A model has been formulated for how Ku and p460 participate in end-joining. To test the model, the specific aims of this proposal are to investigate: 1. The roles of Ku and p460 in the synapsis of DNA ends. A super-large complex of DNA, Ku and p460 was detected and will be characterized to determine if it represents the synapsis of 2 DNA molecules. A 19 Angstrom resolution structure of p460 was obtained, suggesting synapsis may occur in a parallel orientation. Experiments will distinguish between parallel and antiparallel synapsis. 2. The roles of Ku and p460 in the processing of DNA ends. DNA-PK phosphorylation of Ku activates its ATPase activity, which will be tested for possible roles in processing DNA ends: strand exchange, end-unwinding, unwinding coupled to microhomology base pairing, and opening hairpin ends. Ku mutated in its ATP binding motifs will be transfected into Ku mutant cells to test the role of the ATPase in vivo. 3. Later steps in the end-joining reaction. The complex of Ku and p460 on DNA ends was discovered to recruit additional factors. These factors will be identified by protein purification and by testing specific candidates, including XRCC4, DNA ligases I and IV, RPA, HMG1 and HMG2. Once the initial steps have been characterized, a search will be conducted for the complete end-joining reaction in vitro. Ultimately, these studies will lead to a molecular description of both DSB repair and V(D)J recombination, leading to deeper insight into both cancer and the human immune system.