Double Strand Break Repair by Homologous Recombination Is Regulated by Cell Cycle-independent Signaling via ATM in Human Glioma Cells

Abstract

To investigate double strand break (DSB) repair and signaling in human glioma cells, we stably transfected human U87 (ATM ؉ , p53 ؉ ) glioma cells with a plasmid having a single I-SceI site within an inactive green fluorescent protein (GFP) expression cassette, allowing for the detection of homologous recombination repair (HRR) by GFP expression. HRR and nonhomologous end joining (NHEJ) were also determined by PCR. DSB repair was first detected at 12 h postinfection with an adenovirus expressing I-SceI with repair reaching plateau levels between 24 and 48 h. Within this time frame, NHEJ predominated over HRR in the range of 3-50-fold. To assess the involvement of ATM in DSB repair, we first examined whether ATM was associated with the DSB. Chromatin immunoprecipitation showed that ATM was present at the site of the DSB as early as 18 h postinfection. In cells treated with caffeine, an inhibitor of ATM, HRR was reduced, whereas NHEJ was not. In support of this finding, GFP flow cytometry demonstrated that caffeine reduced HRR by 90% under conditions when ATM kinase activity was inhibited. Dominant-negative ATM expressed from adenovirus inhibited HRR by 45%, also having little to no effect on NHEJ. Furthermore, HRR was inhibited by caffeine in serum-starved cells arrested in G 0 /G 1 , suggesting that ATM is also important for HRR outside of the S and G 2 cell cycle phases. Altogether, these results demonstrate that HRR contributes substantially to DSB repair in human glioma cells, and, importantly, ATM plays a critical role in regulating HRR but not NHEJ throughout the cell cycle.

Double-strand break repair by homologous recombination in primary mouse somatic cells requires BRCA1 but not the ATM kinase

“…Mouse models recapitulate many of the phenotypes seen in patients with ATM mutations, including tumor predisposition, radiosensitivity, and gonadal dysgenesis (30,33,34). Although some studies using transformed cell lines have reported that ATM is not required for HDR (44), others have suggested that ATM plays a critical role (45,46). ATM has been reported to be necessary for DSB resection to generate Replication protein A (RPA)-coated ssDNA (29), an essential early intermediate in HDR.…”

Checkpoint response plays a more protective role in HZE particle-irradiated cells than in X ray-irradiated cells

“…Previously, we and others showed that IR-induced checkpoint response mainly promotes HRR and has little effect on NHEJ. [7][8][9][10] These results provide important information that because the NHEJ pathway is inhibited in high LET irradiated cells, HRR might play a more protective role in such cells than that in low LET irradiated cells; because checkpoint facilitates mainly the HRR pathway, checkpoint response might be a more efficient factor for modifying cell sensitivity to high LET IR than to low LET IR. To test this hypothesis, we examined and analyzed the RBE on sensitivity in ATM or ATR, the two most important checkpoint genes, deficient or kinase dead cell lines following either high LET or low LET IR.…”

ATR Affecting Cell Radiosensitivity Is Dependent on Homologous Recombination Repair but Independent of Nonhomologous End Joining

“…This response differs from ATM, another important checkpoint protein that mainly responds to IR but not to UV-induced DNA damage. The role of ATM in cellular radiosensitivity is linked to HRR (11,12), but which pathway, HRR or NHEJ or both, is responsible for the effects of ATR on the sensitivity of cells to IR remains unclear. Our results previously suggested that DNA damage-induced checkpoint facilitates HRR but not NHEJ (7,8).…”