End Joining Reaction in DNA Repair & V(D)J Recombination

描述（由申请人提供）：电离辐射和V（D）J重组产生DNA双链断裂（DSB），通过非同源末端连接（NHEJ）修复。NHEJ所需的蛋白质包括DNA依赖性蛋白激酶（DNA-PK），其由DNA结合亚基（Ku）和催化亚基（DNA-PKcs）组成。目前尚不清楚DNA-PK和其他因子如何将DNA的断裂末端连接在一起，处理末端以进行连接，并通过磷酸化调节反应。长期目标是阐明NHEJ中的每一步，这将有助于更好地了解癌症，免疫和衰老。具体目的是：1.明确NHEJ过程中DNA末端突触形成的机制。初步研究表明，DNA-PKcs在低盐条件下将DNA末端聚集在突触复合物中。建议的研究将研究DNAPKcs自磷酸化对DNA突触的影响，确定在生理盐条件下突触发生所需的因素，并确定突触复合体中DNA-PKcs分子的数量.定义NHEJ过程中如何处理DNA末端进行连接。用于NHEJ的无细胞系统检测到野生型提取物中的有效末端加工和来自Werner综合征（一种过早衰老和癌症的疾病）和Nijmegan断裂综合征（一种辐射敏感性和癌症的疾病）的提取物中的末端加工缺陷。实验将定义相关蛋白质Wm和Nbs 1的作用。末端加工包括模板化核苷酸添加，并且随着dNTPs的添加而增加。实验将确定是否涉及DNA聚合酶。无细胞系统将用于表征V（D）J重组过程中发夹末端的连接，鉴定打开发夹末端的核酸内切酶，并确定DNA-PKcs如何调控发夹的打开。确定NHEJ如何通过蛋白磷酸化调节。为了确定DNA-PKcs激酶活性在NHEJ中的作用，DNA-PKcs突变体提取物将补充活性激酶或渥曼青霉素失活的激酶，并测定DNA末端的加工和连接。实验将确定磷酸酶是否在NHEJ中发挥作用，并确定DNA-PKcs的生理相关靶点。

DESCRIPTION (provided by applicant): Ionizing radiation and V(D)J recombination generate DNA double-strand breaks (DSBs), which are repaired by non-homologous end-joining (NHEJ). Proteins required for NHEJ include DNA-dependent protein kinase (DNA-PK), which consists of a DNA binding subunit (Ku) and a catalytic subunit (DNA-PKcs). It is not yet known how DNA-PK and other factors bring the broken ends of the DNA together, process the ends for joining, and regulate the reaction by phosphorylation. The long-term objective is to elucidate each step in NHEJ, which will lead to a better understanding of cancer, immunity, and aging. The specific aims are:1.To define the mechanism for synapsis of DNA ends during NHEJ. Preliminary studies show that DNA-PKcs brings DNA ends together in a synaptic complex at low salt. Proposed studies will study the effect of DNAPKcs autophosphorylation on DNA synapsis, determine the factors required for synapsis to occur at physiological salt, and determine the number of DNA-PKcs molecules in the synaptic complex.2. To define how the DNA ends are processed for joining during NHEJ. A cell-free system for NHEJ detected efficient end-processing in wild type extracts and deficient end-processing in extracts from Werner's Syndrome, a disease of premature aging and cancer, and Nijmegan Breakage Syndrome, a disease of radiation sensitivity and cancer. Experiments will define the roles of the associated proteins, Wm and Nbs1. End-processing included templated nucleotide addition and increased with addition of dNTPs. Experiments will determine if DNA polymerase(s) are involved. The cell free system will be used to characterize the joining of hairpin ends, which occurs during V(D)J recombination, identify the endonuclease that opens the hairpin ends, and determine how hairpin opening is regulated by DNA-PKcs.3. To define how NHEJ is regulated by protein phosphorylation. To determine the role of DNA-PKcs kinase activity in NHEJ, DNA-PKcs-mutant extracts will be supplemented with the active kinase or wortmannin-inactivated kinase and assayed for processing and joining of DNA ends. Experiments will determine whether phosphatases play a role in NHEJ, and identify physiologically relevant targets of DNA-PKcs.