International Journal of Radiation Oncology*Biology*Physics
Biology contributionMarked Dependence on Growth State of Backup Pathways of NHEJ
Introduction
The DNA double-strand break (DSB) is a highly cytotoxic lesion induced in the genome of cells through a variety of endogenous processes, as well as by external agents such as ionizing radiation (IR) and radiomimetic drugs. In cells of higher eukaryotes, DSBs are repaired mainly by nonhomologous end joining (NHEJ) (1, 2, 3). This mode of DSB repair is homology independent and restores the integrity of the DNA molecule without necessarily preserving its sequence in the vicinity of the break (1, 4, 5).
At the heart of this process, is a serine/threonine protein kinase, specifically activated by DNA ends, that consists of the heterodimeric complex Ku (Ku70 and Ku80 subunits), and the DNA-PK catalytic subunit, DNA-PKcs. Aided by its structure (6), Ku captures DNA ends and recruits DNA-PKcs, which undergoes conformational changes and probably dimerizes to generate a platform for the subsequent processing of the DSB (7). Processing culminates with end ligation catalyzed by DNA ligase IV. We refer to this pathway as D-NHEJ to indicate its dependence on DNA-PK (8, 9).
Cells with mutations in components of D-NHEJ remain capable of repairing the majority of the IR-induced DSBs using an alternative, slower process that, unexpectedly, is not sensitive to mutations in genes required for homologous recombination repair (HRR) (8, 9, 10). We therefore refer to this form of DSB repair as a distinct form of end joining that is normally suppressed by D-NHEJ (11). However, when D-NHEJ is genetically or chemically compromised, this form of end joining acts like a backup and restores the majority of DSBs in the genome, albeit frequently incorrectly. We propose the term B-NHEJ for this form of nonhomologous end joining to differentiate it from D-NHEJ and to indicate the putative backup function (8). Recent work implicates DNA ligase III in B-NHEJ (12, 13) and suggests that the repair module PARP-1/XRCC1/DNA ligase III (13, 14), firmly implicated in the repair of single strand breaks (SSB) (15), also contributes to DSB repair.
D-NHEJ operates efficiently in all phases of the cell cycle with only relatively small fluctuations in the half times between G1, S, and G2 (16, 17). A wider range of differences is observed, however, between exponentially growing and plateau-phase cells, implicating growth state as a parameter in the efficiency of D-NHEJ. Recent studies provide evidence for a direct link between growth state and repair of DNA DSBs by implicating epidermal growth factor receptor (EGFR) signaling to DNA-PK activity (18, 19, 20).
The link between D-NHEJ and growth state raises the question as to whether B-NHEJ is also subject to similar regulation. Here we present data indicating a reduction in the capacity of cells to rejoin DSBs by B-NHEJ as they enter the plateau phase of growth. This reduction in the repair capacity is not due solely to the associated accumulation of cells in G1 phase. The results indicate for the first time linkages between growth state and backup pathways of DSB repair and suggest molecular connections that require further exploration.
Section snippets
Methods and Materials
Spontaneously immortalized fibroblasts from single LIG4+/+/p53–/– and LIG4–/–/p53–/– embryos (abbreviated here as WT [wild type] and LIG4–/– MEFs, respectively; kindly provided by Dr. F. Alt) (21) were grown without further subcloning in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells show no contact inhibition and cannot be maintained for prolonged periods of time in the plateau phase of growth. Floating cells are not included in the analysis of
Results
We conducted control experiments to confirm the genetic background of the LIG4–/– cells. Figure 1A and 1B summarize results of quantitative, real-time PCR carried out with primers that amplify a sequence within the DNA ligase IV active site—the region of the gene that has been deleted in LIG4–/– MEFs (21). Figure 1A shows the real-time PCR results including the associated melting curves, and Fig. 1B shows their quantitative analysis for LIG4–/– and WT cells. As expected, LIG4–/– cells show
Discussion
The results presented here indicate a marked reduction in the efficiency of DSB rejoining in LIG4–/– cells after transition from the exponential into the plateau phase of growth. Because under the same conditions a difference in the ability of WT cells to repair DSBs is not detectable, we conclude that the dependence of B-NHEJ on growth state is stronger than that of D-NHEJ. The reduction in DSB repair efficiency on transition into the plateau phase of growth can derive from the accumulation of
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2020, DNA RepairCitation Excerpt :Ligation is mediated by DNA ligases I and III (LIG1 and LIG3) [33]. Alt-EJ is active throughout the cell cycle (25–27), but shows increased activity in G2-phase [26] and is frequently ablated in G0 cells [34–36]. Alt-EJ is strongly implicated in translocation formation [37] and can generate relatively large deletions at the DSB junction.
DNA double-strand repair by nonhomologous end joining and its clinical relevance
2016, DNA Repair in Cancer Therapy: Molecular Targets and Clinical Applications: Second EditionAlternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations
2015, Mutation Research - Genetic Toxicology and Environmental MutagenesisCitation Excerpt :Indeed, all available evidence points to marked differences in the concepts underpinning DSB management between higher and lower eukaryotes. Differences include the apparently dominant role in the latter of the end-joining pathways described next and the associated difficulty in detecting DSB repair defects under certain conditions in irradiated HRR mutants [24–26]. These phenotypic characteristics together with the documented contribution of HRR to DSB repair as demonstrated by the increased radiosensitivity of HRR-mutants and using γ-H2AX assays [22,27,28], suggest evolutionary divergence in the principles of HRR-utilization, which are further discussed below.
This work was supported by grants from the DFG and Volkswagenstiftung. We thank Tamara Mussfeldt for expert assistance with flow cytometry and centrifugal elutriation.
Conflict of interest: none.
- 1
Dr. Wenqi Wu is currently at the Department of Urology, Minimally Invasive Surgery Center, the First Hospital of Guangzhou Medical College, Guangzhou, China.
- 2
Frank Windhofer, Ph.D., and Wenqi Wu, M.D., contributed equally to this work.