Abstract
The Ku heterodimer (Ku70/Ku80) is the central DNA binding component of the classical non-homologous end joining (NHEJ) pathway that repairs DNA double-stranded breaks (DSBs), serving as the scaffold for the formation of the NHEJ complex. Here we show that Ku70 is phosphorylated on Serine 155 in response to DNA damage. Expression of Ku70 bearing a S155 phosphomimetic substitution (Ku70 S155D) in Ku70-deficient mouse embryonic fibroblasts (MEFs) triggered cell cycle arrest at multiple checkpoints and altered expression of several cell cycle regulators in absence of DNA damage. Cells expressing Ku70 S155D exhibited a constitutive DNA damage response, including ATM activation, H2AX phosphorylation and 53BP1 foci formation. Ku70 S155D was found to interact with Aurora B and to have an inhibitory effect on Aurora B kinase activity. Lastly, we demonstrate that Ku and Aurora B interact following ionizing radiation treatment and that Aurora B inhibition in response to DNA damage is dependent upon Ku70 S155 phosphorylation. This uncovers a new pathway where Ku may relay signaling to Aurora B to enforce cell cycle arrest in response to DNA damage.
Highlights
Double-strand breaks (DSBs) are the most dangerous form of DNA damage, as improperly repaired, they can result in genetic alterations, leading to genomic instability, a hallmark of cancer
We demonstrate that Ku70 S155 is phosphorylated after IR, and show that constitutive expression of the phosphomimetic mutant Ku70 S155 to aspartic acid (S155D) induces a DNA damage response (DDR) marked by a constitutive activation of ATM and cell cycle arrest at both the G1/S and G2/M checkpoints
Substitution to a phosphomimetic residue (S155D) conferred hypersensitivity to IR treatment. These analyses suggested that S155 is a phosphorylation site that is targeted for phosphorylation in response to DNA damage
Summary
Double-strand breaks (DSBs) are the most dangerous form of DNA damage, as improperly repaired, they can result in genetic alterations, leading to genomic instability, a hallmark of cancer. The PI3K-like kinases are the main regulators of the DDR and orchestrate many phosphorylation events at the site of DNA damage that promote DNA repair[3]. Aurora B’s activities, are not restricted to mitosis, as it is expressed throughout the cell cycle and there is evidence that it contributes to G1/S and G2/M checkpoint regulation[6,7] Ectopic expression of both Aurora A and B results in chromosomal abnormalities and cellular transformation, and overexpression of Aurora kinases is observed in a number of different cancers[5,8,9,10]. Following the introduction of a DSB, Ku rapidly binds the broken ends and forms a complex with the PI3K-like kinase DNA-PKcs to recruit other NHEJ repair factors and promote repair of the break[22]. Ku has important functions in telomere maintenance and protection, and Ku-deficiency leads to telomere defects[23,24]
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