Relevant Targets of the DNA Dependent Protein Kinase

描述(申请人提供)：DNA双链断裂的有效修复对于维持高等生物的染色体完整性和持久性至关重要。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还在淋巴细胞发育中发挥作用，以修复VDJ重组过程中发生的链断裂，VDJ重组是一种提供功能性抗原受体基因组装的定点重组过程。如果VDJ重组受损，B和T淋巴细胞发育受阻，导致疾病，严重的联合免疫缺陷[SCID]。在过去的十年里，一项密集的研究工作集中在NHEJ上，导致对DSB是如何解决的有了合理的理解。已鉴定出六种不同的基因产物[Ku70、Ku86、XRCC4、DNA连接酶IV、Artemis和DNA-PKcs]。其中三个包括一个复合体，DNA依赖蛋白激酶(DNA-PK)。这种蛋白质复合体在非同源末端连接过程中是中心的，因为DNA-PK最初识别并结合到受损的DNA上，然后将其他修复活动靶向DNA损伤的部位。尽管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，但原因尚不清楚。DNA-PK改变了NHEJ的一个或多个重要中介(S)的功能，这是拟议的研究中要检验的中心假说。已知的在NHEJ中起作用的六个因子中，有五个是DNA-PK的激酶活性的靶标(无论是在体外还是在体内)；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子中的任何一个的磷酸化在功能上是相关的。这项拟议的研究将定位DNA-PK在这五个因素中的每一个的目标磷酸化位点。使用突变方法，我们将确定每个因子的DNA-PK磷酸化是否在功能上相关。综上所述，由于非同源末端连接途径对于维持基因组的完整性至关重要，因此了解该途径的功能具有无可置疑的意义。然而，如果不清楚DNA-PK的激酶活性激活了什么因子(S)，我们对非同源DNA端连接是如何工作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.