Requirement for the kinase activity of XXXX DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining.

@entity1 @entity535 @entity10321 @entity10320 @entity1919 @entity10322 @entity563 @entity1889 @entity4357
The catalytic subunit of DNA-dependent protein kinase ( @entity10322 ) is an enormous, 470-kDa protein @entity563 / @entity1889 kinase that has homology with members of the @entity4357 (PI) 3-kinase superfamily. This protein contributes to the repair of DNA double-strand breaks (DSBs) by assembling broken ends of DNA molecules in combination with the DNA-binding factors @entity10320 and @entity10321 . It may also serve as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks. This study attempts to better define the role of protein kinase activity in the repair of DNA DSBs. We constructed a contiguous 14-kb @entity1 @entity10322 cDNA and demonstrated that it can complement the DNA DSB repair defects of two mutant cell lines known to be deficient in @entity10322 (M059J and V3). We then created deletion and site-directed mutations within the conserved PI 3-kinase domain of the @entity10322 gene to test the importance of protein kinase activity for DSB rejoining. These @entity10322 mutant constructs are able to express the protein but fail to complement the DNA DSB or V(D)J recombination defects of @entity10322 mutant cells. These results indicate that the protein kinase activity of @entity10322 is essential for the rejoining of DNA DSBs in @entity1 cells. We have also determined a model structure for the @entity10322 kinase domain based on comparisons to the crystallographic structure of a @entity1919 -dependent protein kinase. This structure gives some insight into which @entity535 residues are crucial for the kinase activity in @entity10322 .
