Relevant Targets of the DNA Dependent Protein Kinase

描述（由申请人提供）：DNA双链断裂的有效修复对于维持染色体完整性和高等生物的持久性至关重要。在高等真核生物中，非同源DNA末端连接（NHEJ）是修复这些断裂的主要途径。NHEJ还能促进淋巴细胞修复在VDJ重组过程中发生的链断裂，这是一种位点特异性重组过程，为功能性抗原受体基因的组装提供了条件。如果VDJ重组受损，B淋巴细胞和T淋巴细胞发育受阻，导致疾病，严重的联合免疫缺陷[SCID]。在过去的十年中，对NHEJ进行了深入的研究，从而对dsb的解决方式有了合理的理解。已经鉴定出六种不同的基因产物在该途径中起作用[Ku70， Ku86, XRCC4， DNA连接酶IV， Artemis和DNA- pkcs]。其中三个组成一个复合体，DNA依赖性蛋白激酶（DNA- pk）。该蛋白复合物在非同源末端连接过程中处于中心位置，因为DNA- pk最初识别并结合受损DNA，然后将其他修复活动靶向DNA损伤部位。虽然最近的数据明确表明DNA-PK的激酶活性在NHEJ期间是必不可少的，但尚不清楚原因。DNA-PK改变NHEJ的一种或多种重要介质的功能是本研究有待验证的中心假设。已知在NHEJ中起作用的六个因子中有五个是DNA-PK激酶活性的靶标（无论是在体外还是体内）；只有DNA连接酶IV不是。然而，到目前为止，没有令人信服的证据表明这些因素的磷酸化在功能上是相关的。拟议的研究将绘制DNA-PK在这五个因素中的靶磷酸化位点。使用突变方法，我们将确定每个因子的DNA-PK磷酸化是否与功能相关。总之，由于非同源末端连接途径对于维持基因组完整性至关重要，因此了解该途径的功能是毫无疑问的。然而，如果不清楚哪些因子被DNA- pk的激酶活性激活，我们对非同源DNA末端连接如何起作用的理解将是不完整的。

DESCRIPTION (provided by applicant): Efficient repair of DNA double strand breaks is essential for the maintenance of chromosomal integrity and persistence of higher organisms. In higher eukaryotes, non-homologous DNA end joining (NHEJ) is the primary pathway that repairs these breaks. NHEJ also functions in developing lymphocytes to repair strand breaks that occur during VDJ recombination, the site-specific recombination process that provides for assembly of functional antigen receptor genes. If VDJ recombination is impaired, B and T lymphocyte development is blocked resulting in the disease, severe combined immunodeficiency [SCID]. In the last decade, an intensive research effort has focused on NHEJ resulting in a reasonable understanding of how DSBs are resolved. Six distinct gene products have been identified which function in this pathway [Ku70, Ku86, XRCC4, DNA ligase IV, Artemis, and DNA-PKcs]. Three of these comprise one complex, the DNA dependent protein kinase (DNA-PK). This protein complex is central during non-homologous end joining because DNA-PK initially recognizes and binds to damaged DNA and then targets other repair activities to the site of DNA damage. Though recent data demonstrate unequivocally that DNA-PK's kinase activity is essential during NHEJ, it is not clear why. That DNA-PK alters the function of one or more important mediator(s) of NHEJ is the central hypothesis to be tested in the proposed studies. Five of the six factors known to function in NHEJ are targets of DNA-PK's kinase activity (either in vitro or in vivo); only DNA ligase IV is not. However, to date there is no compelling evidence that phosphorylation of any of these factors is functionally relevant. The proposed research will map DNA-PK's target phosphorylation sites in each of these five factors. Using a mutational approach, we will then determine whether DNA-PK phosphorylation of each factor is functionally relevant. In sum, since the non-homologous end-joining pathway is critical for the maintenance of genomic integrity, understanding how this pathway functions is of unquestionable relevance. However, our understanding of how nonhomologous DNA end joining works will be incomplete without a clear understanding of what factor(s) are activated by DNA-PK's kinase activity.