Abstract
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.
Original language | English (US) |
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Pages (from-to) | 1462-1470 |
Number of pages | 9 |
Journal | International Journal of Radiation Oncology Biology Physics |
Volume | 68 |
Issue number | 5 |
DOIs | |
State | Published - Aug 1 2007 |
Keywords
- Cell cycle
- DNA Double-strand breaks (DSB)
- DNA ligase IV
- Growth state
- Nonhomologous end-joining (NHEJ)
- Radiation
ASJC Scopus subject areas
- Radiation
- Oncology
- Radiology Nuclear Medicine and imaging
- Cancer Research