A mechanism for DNA-PK activation requiring unique contributions from each strand of a DNA terminus and implications for microhomology-mediated nonhomologous DNA end joining

Abstract

DNA-dependent protein kinase (DNA-PK) is an essential component of the nonhomologous end joining pathway (NHEJ), responsible for the repair of DNA double-strand breaks. Ku binds a DSB and recruits the catalytic subunit, DNA-PKcs, where it is activated once the kinase is bound to the DSB. The precise mechanism by which DNA activates DNA-PK remains unknown. We have investigated the effect of DNA structure on DNA-PK activation and results demonstrate that in Ku-dependent DNA-PKcs reactions, DNA-PK activation with DNA effectors containing two unannealed ends was identical to activation observed with fully duplex DNA effectors of the same length. The presence of a 6-base single-stranded extension resulted in decreased activation compared to the fully duplex DNA. DNA-PK activation using DNA effectors with compatible termini displayed increased activity compared to effectors with noncompatible termini. A strand orientation preference was observed in these reactions and suggests a model where the 3′ strand of the terminus is responsible for annealing and the 5′ strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ.

Correlativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells

“…Then, the conformation of Ku changes, allows it to interact with DNA-PKcs [8,9]. After autophosphorylation, DNA-PKcs recruits XRCC4/ligase IV complex to process the DNA ends and initiate religation to form a single DNA molecule [10,11]. Our data shows that expressions of NHEJ protein Ku80 and DNAPKcs were higher than that of HR protein ATM in all eight cell lines and confirmed the above conclusion that NHEJ played the major role in DSB repair.…”

Coordination of DNA–PK Activation and Nuclease Processing of DNA Termini in NHEJ

“…Studies from our lab have revealed that DNA-PK is preferentially activated by 3 0 -pyrimidine-rich sequences, and this supports the theory that single-strand ends of DNA may play an important and differential role in NHEJ catalyzed repair of DSBs (65). Building upon this work we have discovered that DNA-PK is preferentially activated by substrates containing 5 0 -single-strand overhangs compared to 3 0 -single-strand overhangs (66). These combined results suggest that individual DNA termini play an important role in activation of DNA-PK.…”

The pyridinylfuranopyrimidine inhibitor, PI-103, chemosensitizes glioblastoma cells for apoptosis by inhibiting DNA repair

“…In nontransformed cells, DNA-PK is usually but not always (for example, Yavuzer et al, 1998) believed to be directly activated on a DSB (Pawelczak and Turchi, 2008), thereafter initiating the repair machinery while concurrently activating survival pathways, such as PI3K/Akt-mediated signaling (Bozulic et al, 2008), possibly by direct phosphorylation (Feng et al, 2004). In tumor cells, however, which are usually more dependent on PI3K-mediated survival signaling and frequently express stimuli-independent activity of this signaling cascade (Yuan and Cantley, 2008), the relationship between PI3K and DNA-PK can be altered.…”