Biology Contribution
Widespread Dependence of Backup NHEJ on Growth State: Ramifications for the Use of DNA-PK Inhibitors

https://doi.org/10.1016/j.ijrobp.2010.08.018Get rights and content

Purpose

The backup pathway of nonhomologous end joining (B-NHEJ) enables cells to process DNA double-strand breaks (DSBs) when the DNA-PK–dependent pathway of NHEJ (D-NHEJ) is compromised. Our previous results show marked reduction in the activity of B-NHEJ when LIG4–/– mouse embryo fibroblasts (MEFs) cease to grow and enter a plateau phase. The dependence of B-NHEJ on growth state is substantially stronger than that of D-NHEJ and points to regulatory mechanisms or processing determinants that require elucidation. Because the different D-NHEJ mutants show phenotypes distinct in their details, it is necessary to characterize the dependence of their DSB repair capacity on growth state and to explore species-specific responses.

Methods and Materials

DSB repair was measured in cells of different genetic background from various species using pulsed-field gel electrophoresis, or the formation of γ-H2AX foci, at different stages of growth.

Results

Using pulsed-field gel electrophoresis, we report a marked reduction of B-NHEJ during the plateau phase of growth in KU and XRCC4, mouse or Chinese hamster, mutants. Notably, this reduction is only marginal in DNA-PKcs-deficient cells. However, reduced B-NHEJ is also observed in repair proficient, plateau-phase cells after treatment with DNA-PK inhibitors. The reduction of B-NHEJ activity in the plateau phase of growth does not derive from the reduced expression of participating proteins, is detectable by γ-H2AX foci analysis, and leads to enhanced cell killing.

Conclusions

These results further document the marked dependence on growth state of an essential DSB repair pathway and show the general nature of the effect. Molecular characterization of the mechanism underlying this response will help to optimize the administration of DNA repair inhibitors as adjuvants in radiation therapy.

Introduction

During nonhomologous end-joining (NHEJ), broken DNA ends are captured by the Ku heterodimer (Ku70/80) that recruits and activates DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which in turn mediates the ligation of the DNA ends by the DNA Ligase IV/XRCC4/XLF complex 1, 2. DNA polymerases μ and λ, as well as polynucleotide kinase and Artemis, are thought also to be involved in DNA end processing 3, 4, 5. We proposed referring to this pathway as D-NHEJ to indicate its dependence on DNA-PK and to discriminate it from other pathways operating using similar basic principles 4, 5.

Cells with mutations in components of D-NHEJ remain capable of repairing the majority of IR-induced DSBs, albeit with slower kinetics. Unexpectedly, this repair pathway is not sensitive to mutations in genes required for homologous recombination repair (HRR) (6). We therefore proposed referring to this DSB processing as a distinct form of end joining that is normally suppressed by D-NHEJ (7). When D-NHEJ is genetically or chemically compromised, this form of end joining acts as a backup and restores integrity in the genome by repairing the majority of DSBs, albeit frequently incorrectly. We proposed the term B-NHEJ for this form of NHEJ to differentiate it from D-NHEJ and bring to the fore its putative backup function (5). Biochemical studies implicate DNA ligase III in B-NHEJ 8, 9 and suggest that the repair module PARP-1/XRCC1/DNA ligase III 9, 10, which is already known to process single strand breaks (SSBs) (11), also contributes to DSB repair.

D-NHEJ operates efficiently across all phases of the cell cycle 12, 13, 14 and shows only a small dependence on cell growth state and growth factor signaling 15, 16. B-NHEJ, in contrast, shows strong cell cycle dependence and operates more efficiently in G2 (17). In addition, B-NHEJ shows a pronounced dependence on growth state (18). This dependence points to regulatory mechanisms and processing determinants that require elucidation.

Previously, the dependence of B-NHEJ on growth conditions has only been demonstrated for LIG4–/– mouse embryo fibroblasts (MEFs). It has therefore remained unknown whether it reflects an isolated response or a more general phenomenon that can be seen across species and in different D-NHEJ mutants. That this is not a trivial inquiry is indicated by the observation that different D-NHEJ mutants show qualitatively and quantitatively distinct phenotypes (19). The results presented here show that the functional limitation of B-NHEJ under conditions of reduced growth is a general phenomenon that can be observed across species in all D-NHEJ mutants expect DNA-PKcs mutants.

Section snippets

Cells and cell culture conditions

Chinese hamster ovary (CHO), xr-1, XR-C1-3, and xrs-5 cells were cultured in McCoy’s 5A medium supplemented with 10% fetal calf serum and antibiotics. MEFs generated from KU70–/– or KU80–/– mice and human glioma cells (M059K) were grown in D-MEM supplemented with 10% fetal calf serum. For DSB repair analysis, 1–2 × 105 cells were plated in 60-mm dishes. Cell radiosensitivity to killing was determined by clonogenic assay. Flow cytometry was carried out using standard protocols.

Irradiation and pulsed-field gel electrophoresis

Irradiations were

B-NHEJ is compromised in plateau-phase D-NHEJ mutants across species

To examine whether the previously reported (18) marked growth-state effect on B-NHEJ is restricted to LIG4–/– cells, we examined KU80–/–- and KU70–/–-deficient MEFs. Figure 1A shows typical growth curves, and Fig. 1B and 1C show the repair kinetics during exponential growth (Day 2) or in the plateau phase (Day 4), together with typical PFGE gels and the corresponding cell cycle distribution. The dotted line depicts the previously reported (18) repair kinetics of exponentially growing wildtype

Growth state as a determinant of B-NHEJ efficiency

The results presented here extend our previous investigations on the subject (18). The observation that KU70–/– and KU80–/– MEFs, as well as XRCC4-deficient CHO cells, show compromised function of B-NHEJ under conditions of reduced growth, similar in magnitude to that of LIG4–/– MEFs, indicates the general character of the response regardless of the D-NHEJ mutation or the species of origin of the tested cells. The conspicuous and puzzling exception of DNA-PKcs mutants may point to determinants

Acknowledgments

We thank Dr. T. Stamato for providing xr-1 cells, Dr. Malgorzata Z. Zdzienicka for XR-C1-3 cells, Dr. P. Jeggo for Xrs-5 cells, Dr. J. Allalunis-Turner for M059K cells, and Drs. D. Chen and G. Li for Ku80–/– and Ku70–/–mouse embryo fibroblasts.

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      Citation Excerpt :

      Together these studies suggest that A-EJ is active in all the phases of the cell cycle but in contrast to C-NHEJ, it increases as cells enter S-phase, concomitantly with CDK activity (Fig. 2). In addition, growth state also influences A-EJ since IR-induced DSB repair is markedly reduced in plateau-phase cultures of Lig4-null [123] or Ku-null [124] but not wild-type MEFs. In addition serum deprivation impacts on survival of C-NHEJ defective but not wild-type MEFs [125].

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    Authors SKS and WW contributed equally to this work.

    Supported by grants from the Bundes Ministerium fuer Bildung und Forschung (BMBF) and the Deutsche Forschungsgemeinschaft (DFG). Special thanks to Tamara Mussfeldt for expert assistance with flow cytometry and centrifugal elutriation.

    Conflict of interest: none.

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