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The DNA damage response (DDR) is a complex signaling network that leads to damage repair while modulating numerous cellular processes. DNA double-strand breaks (DSBs)-a highly cytotoxic DNA lesion-activate this system most vigorously. The DSB response network is orchestrated by the ATM protein kinase, which phosphorylates key players in its various branches. Proteasome-mediated protein degradation plays an important role in the proteome dynamics following DNA damage induction. Here, we identify the nuclear proteasome activator PA28 gamma (REG gamma; PSME3) as a novel DDR player. PA28 gamma depletion leads to cellular radiomimetic sensitivity and a marked delay in DSB repair. Specifically, PA28 gamma deficiency abrogates the balance between the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair. Furthermore, PA28 gamma is found to be an ATM target, being recruited to the DNA damage sites and required for rapid accumulation of proteasomes at these sites. Our data reveal a novel ATM-PA28 gamma-proteasome axis of the DDR that is required for timely coordination of DSB repair.

The protein kinases ATM and DNA-PKcs play critical roles in the cellular response to DNA double strand breaks (DSBs). ATM and DNA-PKcs are activated in response to DSBs and play several important roles in propagation of the damage signal and for the repair of DNA damage. Recent work from several groups, including ours, has focused on studying the dynamics of each of these proteins at DSBs and the requirements and factors which play a role(s) in this process. The use of live cell imaging of fluorescently-tagged ATM and DNA-PKcs has allowed us to study the real-time response of these proteins to laser-generated DNA damage in vivo. Here, we will extensively discuss the behavior of the ATM and DNA-PKcs proteins at DSB sites.

The DNA damage response (DDR) is a complex signaling network that is induced by DNA lesions and vigorously activated by double strand breaks (DSBs). The DSB response is mobilized by the nuclear protein kinase ATM, which phosphorylates key players in its various branches. SFPQ (PSF) and NONO (p54) are nuclear proteins that interact with each other and have diverse roles in nucleic acids metabolism. The SFPQ/NONO heterodimer was previously found to enhance DNA strand break rejoining in vitro. Our attention was drawn to these two proteins as they interact with the nuclear matrix protein Matrin 3 (MATR3), which we found to be a novel ATM target. We asked whether SFPQ and NONO too are involved in the DSB response. Proteins that function at the early phase of this response are often recruited to the damaged sites. We observed rapid recruitment of SFPQ/NONO to sites of DNA damage induced by laser microbeam. In MATR3 knockdown cells SFPQ/NONO retention at DNA damage sites was prolonged. SFPQ and MATR3 depletion led to abnormal accumulation of cells at the S-phase of the cell cycle following treatment with the radiomimetic chemical neocarzinostatin. Notably, proteins involved in DSB repair via nonhomologous end-joining co-immunoprecipitated with NONO; SFPQ depletion delayed DSB repair. Collectively the data suggest that SFPQ, NONO and MATR3 are involved in the early stage of the DSB response, setting the scene for DSB repair.

High linear energy transfer (LET) ionizing radiation induces complex DNA damage that consists of multiple DNA lesions, including abasic sites, damaged bases, single-strand breaks, and double-strand...

Reversible phosphorylation is an essential posttranslational modification to turn on/off a protein function and to regulate many cellular activities, including DNA repair. A DNA double-strand break (DSB) is the most lethal form of DNA damage and is mainly fixed by the error-prone nonhomologous end joining (NHEJ)-mediated repair and by the high-fidelity homology recombination (HR)-mediated repair. We found previously that protein phosphatase PP4 is required for HR-mediated DSB repair. In this report, we showed that depletion of PP4C by siRNA compromised NHEJ-mediated repair of DSBs induced by the nuclease I-SceI. Both PP4C and its regulatory subunit PP4R2 physically interacted with the chromatin condensation factor KAP1 (KRAB-associated protein 1). Depletion of PP4C led to sustained phosphorylation of KAP1 at Ser824. Conversely, overexpression of PP4C resulted in a decrease of KAP1 phosphorylation. PP4 dephosphorylated pKAP1 in vitro. Inhibition of KAP1 expression resulted in a defect on NHEJ-mediated DSB repair, and co-depletion of PP4c and KAP1 did not have significant synergistic effect on NHEJ-mediated DSB repair. Taken together, our results suggest that PP4C and KAP1 are in the same epistasis group, and PP4 is involved in NHEJ-mediated DSB repair, possibly through regulating the phosphorylation status of KAP1.