Marked dependence on growth state of backup pathways of NHEJ.
OBJECTIVE
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
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 IIIalpha.


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.
Institute of Medical Radiation Biology, University Duisburg-Essen Medical School, Essen, Germany.
Frank
F
Windhofer
Wenqi
W
Wu
Minli
M
Wang
Satyendra K
SK
Singh
Janapriya
J
Saha
Bustanur
B
Rosidi
George
G
Iliakis
D000818:Animals; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000172:deficiency / Q000502:physiology*; D004260:DNA Repair / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000502:physiology*; D016193:G1 Phase; D051379:Mice; D000075223:Poly-ADP-Ribose Binding Proteins; D029867:Xenopus Proteins
D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; C519508:XLF protein, mouse; D029867:Xenopus Proteins; D011088:DNA Ligases; D000072481:DNA Ligase ATP; C000618816:DNA ligase III alpha protein, Xenopus; C000618818:Lig3 protein, mouse