Marked Dependence on Growth State of Backup Pathways of NHEJ

Frank Windhofer, Wenqi Wu, Minli Wang, Satyendra K. Singh, Janapriya Saha, Bustanur Rosidi, George Iliakis

Research output: Contribution to journalArticle

35 Scopus citations

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 languageEnglish (US)
Pages (from-to)1462-1470
Number of pages9
JournalInternational Journal of Radiation Oncology Biology Physics
Volume68
Issue number5
DOIs
StatePublished - 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

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