The role of DNA dependent protein kinase in synapsis of DNA ends.
DNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks the action of exonucleases and ligases. The DNA termini become accessible after autophosphorylation of DNA-PK(CS), which we demonstrate to require synapsis of DNA ends. Interestingly, the presence of DNA-PK prevents ligation of the two synapsed termini, but allows ligation to another DNA molecule. This alteration of the ligation route is independent of the type of ligase that we used, indicating that the intrinsic architecture of the DNA-PK complex itself is not able to support ligation of the synapsed DNA termini. We present a working model in which DNA-PK creates a stable molecular bridge between two DNA ends that is remodeled after DNA-PK autophosphorylation in such a way that the extreme termini become accessible without disrupting synapsis. We infer that joining of synapsed DNA termini would require an additional protein factor.
Weterings
Eric
E
Department of Cell Biology and Genetics, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Verkaik
Nicole S
NS
Brüggenwirth
Hennie T
HT
Hoeijmakers
Jan H J
JH
van Gent
Dik C
DC
D000255:Adenosine Triphosphate / Q000378:metabolism; D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D018983:DNA Footprinting; D011088:DNA Ligases / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D008954:Models, Biological; D009687:Nuclear Proteins; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011995:Recombination, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D011088:DNA Ligases