Unraveling the Complexities of DNA-Dependent Protein Kinase Autophosphorylation

Abstract

f DNA-dependent protein kinase (DNA-PK) orchestrates DNA repair by regulating access to breaks through autophosphorylations within two clusters of sites (ABCDE and PQR). Blocking ABCDE phosphorylation (by alanine mutation) imparts a dominant negative effect, rendering cells hypersensitive to agents that cause DNA double-strand breaks. Here, a mutational approach is used to address the mechanistic basis of this dominant negative effect. Blocking ABCDE phosphorylation hypersensitizes cells to most types of DNA damage (base damage, cross-links, breaks, and damage induced by replication stress), suggesting that DNA-PK binds DNA ends that result from many DNA lesions and that blocking ABCDE phosphorylation sequesters these DNA ends from other repair pathways. This dominant negative effect requires DNA-PK's catalytic activity, as well as phosphorylation of multiple (non-ABCDE) DNA-PK catalytic subunit (DNA-PKcs) sites. PSIPRED analysis indicates that the ABCDE sites are located in the only contiguous extended region of this huge protein that is predicted to be disordered, suggesting a regulatory role(s) and perhaps explaining the large impact ABCDE phosphorylation has on the enzyme's function. Moreover, additional sites in this disordered region contribute to the ABCDE cluster. These data, coupled with recent structural data, suggest a model whereby early phosphorylations promote initiation of nonhomologous end joining (NHEJ), whereas ABCDE phosphorylations, potentially located in a "hinge" region between the two domains, lead to regulated conformational changes that initially promote NHEJ and eventually disengage NHEJ.

TNKS1BP1 functions in DNA double-strand break repair though facilitating DNA-PKcs autophosphorylation dependent on PARP-1

“…The autophosphorylation of DNA-PKcs/S2056 can be induced by ionizing radiation and other DNA damage agents [4, 7, 37], and is necessary for the activation of the DSB NHEJ repair pathway. Our study demonstrated that overexpression of TNKS1BP1 effectively induced the autophosphorylation of DNA-PKcs/S2056.…”

“…The DNA dependent protein kinase complex (DNA-PK) is a critical component of the NHEJ pathway, which consists of a Ku70/Ku80 heterodimer and the DNA dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK plays an essential role in the pathway of NHEJ repair, including initiating DSB recognition, regulating access to breaks, promoting repair machinery assembly and DNA molecule ligation [3, 4]. As the catalytic subunit, DNA-PKcs is a member of the phosphatidylinositol-3 (PI-3) kinase-like family (PI3KK), and it is recruited to DNA damage sites by the Ku70/Ku86 heterodimer [5, 6].…”

“…As the catalytic subunit, DNA-PKcs is a member of the phosphatidylinositol-3 (PI-3) kinase-like family (PI3KK), and it is recruited to DNA damage sites by the Ku70/Ku86 heterodimer [5, 6]. The activation of DNA-PKcs in response to DNA damage is dependent on its autophosphorylation within two cluster sites (ABCDE and PQR) [4, 7] and/or phosphorylated by ATM [8]. The activated DNA-PKcs can phosphorylate a series of canonical repair factors in the NHEJ pathway, including Ku70/Ku86, Artemis, XRCC4, DNA ligase IV, XLF, Werner (WRN) and other DNA damage response proteins: including polynucleotide kinase/phosphatase (PNKP), histone H2AX, P53, CHK2, AKT [3, 9].…”

The Novel, Small-Molecule DNA Methylation Inhibitor SGI-110 as an Ovarian Cancer Chemosensitizer

“…DNA (30–50 µg) was hydrolyzed overnight in 1% nitric acid at 70°C in 500µL total volume. Samples were then diluted to 1.5mL final volume in 1% nitric acid and analyzed by ICP-mass spectrometry as we have previously described (24). Briefly, a benchtop series Thermo ICPMS X-series II system with collision cell technology capability and PlasmaLab software were used to quantify CDDP concentrations.…”

XRCC4/XLF Interaction Is Variably Required for DNA Repair and Is Not Required for Ligase IV Stimulation

“…Cells deficient in various c-NHEJ factors are more sensitive to agents that induce replication stress than c-NHEJ-proficient cells (27,28,53). Schwartz et al have shown previously that fibroblasts derived from XLF-deficient patients are sensitive to low-dose aphidicolin, display increased fragile site instability, and display cell cycle disruptions consistent with an inability to resolve DNA damage associated with replication stress (16).…”