Biology contribution
Marked Dependence on Growth State of Backup Pathways of NHEJ

https://doi.org/10.1016/j.ijrobp.2007.04.038Get rights and content

Purpose: Backup pathways of nonhomologous end joining (B-NHEJ) enable cells to repair DNA double-strand breaks (DSBs) when DNA-PK–dependent NHEJ (D-NHEJ) is compromised. Recent evidence implicates growth signaling in the regulation of D-NHEJ. This study was intended to determine whether the ability to repair DSBs by B-NHEJ also depends on growth state.

Methods and Materials: LIG4–/– and wild type (WT) mouse embryo fibroblasts (MEFs) were used. Repair of DSBs was measured by pulsed-field agarose gel electrophoresis. G1 cells were selected by centrifugal elutriation. A plasmid assay was used to measure DNA end-joining activity in whole cell extracts.

Results: Wild-type MEFs efficiently repaired DSBs by D-NHEJ in either the exponential or plateau phase of growth. Because of their defect in ligase IV, which compromises D-NHEJ, LIG4–/– MEFs showed reduced repair capacity but were slowly able to rejoin a large proportion of DSBs via B-NHEJ. B-NHEJ was markedly reduced in the plateau phase of growth or at high radiation doses. Elutriated G1 cells from exponentially growing or plateau-phase LIG4–/– cultures showed a response similar to nonelutriated cells, ruling out that the effect simply reflects redistribution in the cell cycle. An in vitro assay, gauging the activity of B-NHEJ, showed a reduction in DNA end joining during the plateau phase that could be corrected by recombinant DNA ligase IIIα.

Conclusions: Suppression of growth signaling markedly compromises DSB repair by B-NHEJ. This effect is associated with a reduction in DNA ligase III mediated DNA end joining.

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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    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.

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