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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
7R01CA054326-05
Application #
2458069
Study Section
Molecular Biology Study Section (MBY)
Project Start
1992-07-20
Project End
2000-07-31
Budget Start
1997-08-07
Budget End
1998-07-31
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Immune Disease Institute, Inc.
Department
Type
DUNS #
115524410
City
Boston
State
MA
Country
United States
Zip Code
02115
Ranganathan, V; Heine, W F; Ciccone, D N et al. (2001) Rescue of a telomere length defect of Nijmegen breakage syndrome cells requires NBS and telomerase catalytic subunit. Curr Biol 11:962-6
Jin, S; Kharbanda, S; Mayer, B et al. (1997) Binding of Ku and c-Abl at the kinase homology region of DNA-dependent protein kinase catalytic subunit. J Biol Chem 272:24763-6
Boubnov, N V; Wills, Z P; Weaver, D T (1995) Coding sequence composition flanking either signal element alters V(D)J recombination efficiency. Nucleic Acids Res 23:1060-7
Boubnov, N V; Weaver, D T (1995) scid cells are deficient in Ku and replication protein A phosphorylation by the DNA-dependent protein kinase. Mol Cell Biol 15:5700-6
Banga, S S; Hall, K T; Sandhu, A K et al. (1994) Complementation of V(D)J recombination defect and X-ray sensitivity of scid mouse cells by human chromosome 8. Mutat Res 315:239-47
Staunton, J E; Weaver, D T (1994) scid cells efficiently integrate hairpin and linear DNA substrates. Mol Cell Biol 14:3876-83