In the previous grant period the investigator has been studying the scid mutation which affects double strand break repair and VDJ recombination. He has shown that human chromosome 8 complements the defects of the scid mutant and that scid cells are defective in DNA-PKcs. This is an important finding, as one of the major criticisms of the past review was that there was no definitive evidence that scid is DNA-PKcs. In the next finding period the investigator proposes to characterize the mouse DNA-PKcs gene by obtaining the complete cDNA of approximately 12 kb and determining the primary sequence. With this information in hand he will then determine the mouse XRCC7 mutations. Other studies will show whether the XRCC7 mutant are null or missense mutants. The last objective of this first series of experiments will be to complement the scid phenotypes of XRCC7 cells with the DNA-PK cDNA.
A second aim i s to determine the phenotypes of null and kinase-defective DNA-PKcs mutations and see whether they differ. For this, specific mutations in the PI3 kinase domain of DNA-PKcs will be made. Functional domain studies of the DNA-PKcs gene will show which region associates with Ku. This will be done through deletion and insertion mutations constructed in vitro. Each derivative will be tested for DNA repair and VDJ joining activities after transfection into scid cells. They will also be used to express recombinant protein. The purified proteins will be tested for kinase activity and Ku binding. Some of the kinase- mutants may have a dominant negative phenotype. This will be tested by transfection of the mutant derivatives into wildtype mouse cells. DNA-PK targets will be tested directly. The two that will be examined are Ku and RPA. In vivo and in vitro experiments using the mutant DNA-PKcs cells or purified recombinant protein are planned. scid cells will be examined for the ability to repair a specific double strand break. A construct of a duplicated neo gene, one of which is interrupted by a I-Sce I site, will be introduced into the genome. Cutting at the I-Sce I site will be controlled by expression of the I-Sce gene. Repair of the break normally occurs by homologous recombination of some sort. The last aim describes a brief series of experiments to ask whether DNA-PK has some role in regulating S phase. Scid cells will be examined for the ability to delay S following irradiation.