Enhanced fidelity for rejoining radiation-induced DNA double-strand breaks in the G2 phase of Chinese hamster ovary cells

TY - JOUR

T1 - Enhanced fidelity for rejoining radiation-induced DNA double-strand breaks in the G2 phase of Chinese hamster ovary cells

AU - Krüger, Ines

AU - Rothkamm, Kai

AU - Löbrich, Markus

PY - 2004

Y1 - 2004

N2 - The influence of cell cycle phase on the fidelity of DNA double-strand break (DSB) repair is largely unknown. We investigated the rejoining of correct and incorrect DSB ends in synchronized populations of Chinese hamster ovary cells irradiated with 80 Gy X-rays. A specialized pulsed-field gel electrophoresis assay based on quantitative Southern hybridization of individual large restriction fragments was employed to measure correct DSB rejoining by monitoring restriction fragment reconstitution. Total DSB repair, representing both correct and incorrect rejoining, was analyzed using conventional pulsed-field gel electrophoresis. We present evidence that restriction fragment reconstitution is more efficient in G2 than in G1, suggesting that DSB rejoining in G2 proceeds with higher fidelity. DNA-dependent protein kinase-deficient V3 and xrs-6 cells show impaired restriction fragment reconstitution in G1 and G2 compared with wild-type AA8 and K1 cells, demonstrating that the enhanced fidelity of DSB rejoining in G2 occurs by non- homologous end joining. Additionally, homologous recombination-deficient irs1SF and wild-type cells show identical DSB rejoining in G1 and G2. We propose that structural characteristics of G2 phase chromatin, such as the cohesion of sister chromatids in replicated chromatin, limit the mobility of radiation-induced break ends and enhance the fidelity of DSB rejoining.

AB - The influence of cell cycle phase on the fidelity of DNA double-strand break (DSB) repair is largely unknown. We investigated the rejoining of correct and incorrect DSB ends in synchronized populations of Chinese hamster ovary cells irradiated with 80 Gy X-rays. A specialized pulsed-field gel electrophoresis assay based on quantitative Southern hybridization of individual large restriction fragments was employed to measure correct DSB rejoining by monitoring restriction fragment reconstitution. Total DSB repair, representing both correct and incorrect rejoining, was analyzed using conventional pulsed-field gel electrophoresis. We present evidence that restriction fragment reconstitution is more efficient in G2 than in G1, suggesting that DSB rejoining in G2 proceeds with higher fidelity. DNA-dependent protein kinase-deficient V3 and xrs-6 cells show impaired restriction fragment reconstitution in G1 and G2 compared with wild-type AA8 and K1 cells, demonstrating that the enhanced fidelity of DSB rejoining in G2 occurs by non- homologous end joining. Additionally, homologous recombination-deficient irs1SF and wild-type cells show identical DSB rejoining in G1 and G2. We propose that structural characteristics of G2 phase chromatin, such as the cohesion of sister chromatids in replicated chromatin, limit the mobility of radiation-induced break ends and enhance the fidelity of DSB rejoining.

KW - Animals

KW - Blotting, Southern

KW - CHO Cells

KW - Chromatin/chemistry

KW - Cricetinae

KW - DNA/chemistry

KW - DNA Damage/radiation effects

KW - DNA Repair/radiation effects

KW - DNA-Activated Protein Kinase

KW - DNA-Binding Proteins

KW - Female

KW - G1 Phase/radiation effects

KW - G2 Phase/radiation effects

KW - Mutation/genetics

KW - Protein-Serine-Threonine Kinases/deficiency

KW - X-Rays

U2 - 10.1093/nar/gkh586

DO - 10.1093/nar/gkh586

M3 - SCORING: Journal article

C2 - 15148355

VL - 32

SP - 2677

EP - 2684

JO - NUCLEIC ACIDS RES

JF - NUCLEIC ACIDS RES

SN - 0305-1048

IS - 9

ER -