Processing of DNA double strand breaks by alternative non-homologous end-joining in hyperacetylated chromatin

siRNA-mediated knockdown of HDAC2 fails to provoke histone hyperacetylation in Lig4-/- MEFs and has no detectable effect on B-NHEJ function. Treatment with TSA that inhibits multiple HDACs causes efficient, reversible chromatin hyperacetylation in Lig4-/- MEFs, as well as in human HCT116 Lig4-/- cells and the human glioma cell line M059K. The IR yield of DSBs in TSA-treated cells remains similar to that of untreated cells despite the expected chromatin relaxation. In addition, chromatin hyperacetylation leaves unchanged repair of DSBs by B-NHEJ in irradiated exponentially growing, or plateau-phase cells. Notably, under the experimental conditions employed here, chromatin hyperacetylation fails to detectably modulate B-NHEJ in M059K cells as well.In summary, the results show that chromatin acetylation or deacetylation does not affect the kinetics of alternative NHEJ in all types of cells examined both in exponentially growing and serum deprived cultures. We conclude that parameters beyond chromatin acetylation determine B-NHEJ efficiency in the plateau-phase of growth.It is commonly believed that DSBs induced in the genome of higher eukaryotes by widely diverse endogenous and exogenous factors and processes are mainly repaired by non-homologous end-joining (NHEJ) [1-3]. The canonical and widely investigated pathway of NHEJ (D-NHEJ) starts with the binding to the generated ends of the Ku70/Ku80 complex, which then helps recruit the DNA-dependent protein kinase (DNA-PK) as well as other factors, including the nuclease Artemis and the Lig4/Xrcc4/XLF complex. End-joining occurs rapidly, with only minimal processing of the DNA ends to render them ligatable and limited polymerization [2].When D-NHEJ fails, locally in repair proficient cells, and globally in mutants with defects in D-NHEJ components, or in cells treated with DNA-PK inhibitors, an alternative form of end joining operating as backup to D-NHEJ becomes activated (B-NHEJ) [1,4-6]. B-NHEJ utilizes Lig3 and Parp1