DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining

Abstract

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity

“…The assembly and activation of DNA-PK complex are DNA-dependent [ 46 ] on DNA, and they are intricately regulated by multiple post-translation modifications on its components, including DNA-PKcs and Ku70/80 [ 14 , 23 , 24 ]. SIRT6, a deacetylase, dynamically associates with chromatin to facilitate the mobilization of DNA-PKcs onto chromatin following DNA damage.…”

“…It also stabilizes DNA-dependent protein kinase (DNA-PK) at DSBs [ 23 ]. SIRT2 can also interact with DNA-PKcs to facilitate DNA-PKcs interaction with Ku and subsequent localization to DSBs [ 24 ]. Here, we revealed that the DNA-PKcs K525 residue located in the DNA-binding domain is a critical site for crotonylation and is a factor that positively regulates DNA-PKcs DNA-binding activity and DNA-PKcs assembly.…”

“…DNA-PKcs interfaces with Ku at multiple sites within its N-HEAT (1–872 aa) and M-HEAT domains (890–2580 aa). As mentioned above, SIRT2-mediated DNA-PKcs deacetylation promotes the interaction between DNA-PKcs and KU70/80 heterodimer, as well as the assembly of DNA-PK [ 24 ]. Previous studies have shown that direct binding of DNA-PK to DNA facilitates the activation of DNA-PK, and in vitro, DNA-PKcs can bind directly to DNA in a manner of independent of KU dimer [ 9 , 47 ].…”

“…SIRT6 stabilizes DNA-PKcs on chromatin while promoting the function of the latter NHEJ [ 23 ]. The deacetylation of DNA-PKcs by SIRT2 can active DNA-PKcs, consequently enhancing the interaction between DNA-PKcs and KU70/80 heterodimer [ 24 ].…”

Quercetin inhibits necroptosis in cardiomyocytes after ischemia–reperfusion via DNA‐PKcs‐SIRT5‐orchestrated mitochondrial quality control

“…The sirtuin family has a modulatory effect on DNA-PKcs. SIRT2 deacetylase activity enhances the interaction between DNA-PKcs and Ku, localizing double-strand breaks (DSBs) in the DNA and activating DNA-PK, resulting in the phosphorylation of downstream non-homologous end-joining (NHEJ) substrates (Head et al, 2023). In the absence of DNA-PKcs, BI-1 is degraded, which confers resistance to oxidative stress induced by ionizing or high-energy radiation (Zhou et al, 2020).…”