Visualization of a DNA-PK/PARP1 complex.
The DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are critical enzymes that reduce genomic damage caused by DNA lesions. They are both activated by DNA strand breaks generated by physiological and environmental factors, and they have been shown to interact. Here, we report in vivo evidence that DNA-PK and PARP1 are equally necessary for rapid repair. We purified a DNA-PK/PARP1 complex loaded on DNA and performed electron microscopy and single particle analysis on its tetrameric and dimer-of-tetramers forms. By comparison with the DNA-PK holoenzyme and fitting crystallographic structures, we see that the PARP1 density is in close contact with the Ku subunit. Crucially, PARP1 binding elicits substantial conformational changes in the DNA-PK synaptic dimer assembly. Taken together, our data support a functional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair. We also propose a NHEJ model where protein-protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions.
Cancer Research UK DNA Repair Enzymes Group, The Institute of Cancer Research, London SW3 6JB, UK. laura.spagnolo@ed.ac.uk
Laura
L
Spagnolo
Jody
J
Barbeau
Nicola J
NJ
Curtin
Edward P
EP
Morris
Laurence H
LH
Pearl
D000818:Animals; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure*; D004268:DNA-Binding Proteins / Q000502:physiology; D019281:Dimerization; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C499320:PARP1 protein, human; C499321:Parp1 protein, mouse; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; C497264:Prkdc protein, mouse