Poly(ADP-Ribose) polymerase-1 and DNA-dependent protein kinase have equivalent roles in double strand break repair following ionizing radiation.
OBJECTIVE
Radiation-induced DNA double strand breaks (DSBs) are predominantly repaired by nonhomologous end joining (NHEJ), involving DNA-dependent protein kinase (DNA-PK). Poly(ADP-ribose) polymerase-1 (PARP-1), well characterized for its role in single strand break repair, may also facilitate DSB repair. We investigated the activation of these enzymes by differing DNA ends and their interaction in the cellular response to ionizing radiation (IR).


METHODS
The effect of PARP and DNA-PK inhibitors (KU-0058684 and NU7441) on repair of IR-induced DSBs was investigated in DNA-PK and PARP-1 proficient and deficient cells by measuring gammaH2AX foci and neutral comets. Complementary in vitro enzyme kinetics assays demonstrated the affinities of DNA-PK and PARP-1 for DSBs with varying DNA termini.


RESULTS
DNA-PK and PARP-1 both promoted the fast phase of resolution of IR-induced DSBs in cells. Inactivation of both enzymes was not additive, suggesting that PARP-1 and DNA-PK cooperate within the same pathway to promote DSB repair. The affinities of the two enzymes for oligonucleotides with blunt, 3' GGG or 5' GGG overhanging termini were similar and overlapping (K(d)(app) = 2.6-6.4nM for DNA-PK; 1.7-4.5nM for PARP-1). DNA-PK showed a slightly greater affinity for overhanging DNA and was significantly more efficient when activated by a 5' GGG overhang. PARP-1 had a preference for blunt-ended DNA and required a separate factor for efficient stimulation by a 5' GGG overhang.


CONCLUSIONS
DNA-PK and PARP-1 are both required in a pathway facilitating the fast phase of DNA DSB repair.
Mitchell
Jody
J
Northern Institute for Cancer Research, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom.
Smith
Graeme C M
GC
Curtin
Nicola J
NJ
D000818:Animals; D002867:Chromones / Q000494:pharmacology; D020552:Comet Assay; D004247:DNA / Q000528:radiation effects*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000502:physiology*; D004789:Enzyme Activation; D005347:Fibroblasts; D006657:Histones / Q000032:analysis; D006801:Humans; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009841:Oligonucleotides / Q000235:genetics; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*
C499693:8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one; D002867:Chromones; C468783:H2AX protein, human; D006657:Histones; D009025:Morpholines; D009841:Oligonucleotides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D004247:DNA; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase