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) |
|---|---|
| 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 |
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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
Cite this
Marked Dependence on Growth State of Backup Pathways of NHEJ. / Windhofer, Frank; Wu, Wenqi; Wang, Minli; Singh, Satyendra K.; Saha, Janapriya; Rosidi, Bustanur; Iliakis, George.
In: International Journal of Radiation Oncology Biology Physics, Vol. 68, No. 5, 01.08.2007, p. 1462-1470.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Marked Dependence on Growth State of Backup Pathways of NHEJ
AU - Windhofer, Frank
AU - Wu, Wenqi
AU - Wang, Minli
AU - Singh, Satyendra K.
AU - Saha, Janapriya
AU - Rosidi, Bustanur
AU - Iliakis, George
PY - 2007/8/1
Y1 - 2007/8/1
N2 - 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.
AB - 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.
KW - Cell cycle
KW - DNA Double-strand breaks (DSB)
KW - DNA ligase IV
KW - Growth state
KW - Nonhomologous end-joining (NHEJ)
KW - Radiation
UR - http://www.scopus.com/inward/record.url?scp=34547403092&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34547403092&partnerID=8YFLogxK
U2 - 10.1016/j.ijrobp.2007.04.038
DO - 10.1016/j.ijrobp.2007.04.038
M3 - Article
C2 - 17674976
AN - SCOPUS:34547403092
VL - 68
SP - 1462
EP - 1470
JO - International Journal of Radiation Oncology Biology Physics
JF - International Journal of Radiation Oncology Biology Physics
SN - 0360-3016
IS - 5
ER -