Ku Regulates Signaling to DNA Damage Response Pathways through the Ku70 von Willebrand A Domain

Abstract

The Ku heterodimer (Ku70/Ku80) is a main component of the nonhomologous end-joining (NHEJ) pathway that repairs DNA double-strand breaks (DSBs). Ku binds the broken DNA end and recruits other proteins to facilitate the processing and ligation of the broken end. While Ku interacts with many proteins involved in DNA damage/repair-related functions, few interactions have been mapped to the N-terminal von Willebrand A (vWA) domain, a predicted protein interaction domain. The mutagenesis of Ku70 vWA domain S155/D156 unexpectedly increased cell survival following ionizing radiation (IR) treatment. DNA repair appeared unaffected, but defects in the activation of apoptosis and alterations in the DNA damage signaling response were identified. In particular, Ku70 S155A/D156A affected the IR-induced transcriptional response of several activating transcription factor 2 (ATF2)-regulated genes involved in apoptosis regulation. ATF2 phosphorylation and recruitment to DNA damage-induced foci was increased in Ku70-deficient cells, suggesting that Ku represses ATF2 activation. Ku70 S155A/D156A substitutions further enhanced this repression. S155A substitution alone was sufficient to confer enhanced survival, whereas alteration to a phosphomimetic residue (S155D) reversed this effect, suggesting that S155 is a phosphorylation site. Thus, these findings infer that Ku links signals from the DNA repair machinery to DNA damage signaling regulators that control apoptotic pathways.

The Ku heterodimer: Function in DNA repair and beyond

“…Overall, the function of this domain is not well characterized but there is emerging evidence that it has an important role in DSB repair and telomere regulation. In Saccaromyces cerevisae, mutations in helix 5 of Ku80 abrogated telomere silencing, while mutations in helix 5 of Ku70 had a negative impact on DSB repair in yeast and mouse cells [19][20][21]. Specific residues in the Ku70 vWA domain were also shown to be critically implicated in conferring abasic site processing by Ku in vitro [22,23].…”

“…Specific residues in the Ku70 vWA domain were also shown to be critically implicated in conferring abasic site processing by Ku in vitro [22,23]. Additionally, another region of the human Ku70 vWA domain in helix 4 was found to regulate DNA damage signaling to apoptosis [21]. Consistent with its predicted role as a protein-protein interaction surface, the vWA domain has been shown to mediate an interaction with both the telomere complex component telomere repeat binding factor 2 (TRF2) and NHEJ factor aprataxin and PNKP like factor (APLF) [20,24].…”

“…Furthermore, Ku80 null cells were shown to have increased S phase inhibition after DNA damage due to increased ATM activity [136]. We identified a specific residue in Ku70's vWA domain, serine 155, that when mutated to alanine results in decreased DNA damage signaling and apoptotic activation after IR, despite having no impact on Ku's ability to function in C-NHEJ [21]. These results suggest that in addition to its essential role in DNA repair, modification of Ku also signals to the ATM dependent DDR cascade to regulate cell fate after DNA damage.…”

“…Despite these observations, there has been little insight into the precise mechanism of Ku end bridging. A mutation in helix 5 of the Ku70 vWA domain, noted for its ability to impair DSB repair in both yeast and mammalian cells, was found to decrease multimerization of Ku proteins, leading to the speculation that this helix mediates DNA end bridging through the binding of two Ku70 molecules [19][20][21].…”

Ku70 and non-homologous end joining protect testicular cells from DNA damage

“…Furthermore, Ku70 seems to play a role in apoptosis regulation, possibly through its interaction with BAX (Amsel et al, 2008;Harrington et al, 1992) and by influencing the cell survival transcription network (Fell and Schild-Poulter, 2012). In all, absence of Ku in metaphase cells could lead to apoptosis by pleiotropic effects influencing DNA damage response, SAC pathways and survival networks.…”