Radiation-induced genomic rearrangements formed by nonhomologous end-joining of DNA double-strand breaks

TY - JOUR

T1 - Radiation-induced genomic rearrangements formed by nonhomologous end-joining of DNA double-strand breaks

AU - Rothkamm, K

AU - Kühne, M

AU - Jeggo, P A

AU - Löbrich, M

PY - 2001/5/15

Y1 - 2001/5/15

N2 - Two major pathways for repairing DNA double-strand breaks (DSBs) have been identified in mammalian cells, nonhomologous end-joining (NHEJ) and homologous recombination (HR). Inactivation of NHEJ is known to lead to an elevated level of spontaneous and radiation-induced chromosomal rearrangements associated with an increased risk of tumorigenesis. This has raised the idea of a caretaker role for NHEJ. It is, however, not known whether NHEJ itself can also cause rearrangements. To investigate, on the DNA level, the influence of a defect in NHEJ on the formation of genomic rearrangements, we applied an assay based on Southern hybridization that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionizing radiation. After 80 Gy of X-irradiation at a high dose rate (23 Gy/min), wild-type cells repaired 50% of the induced DSBs within 24 h by incorrect rejoining. This frequency of DSB misrejoining is considerably reduced in NHEJ-deficient cells. Low-dose-rate experiments, in which the cells were exposed to 80 Gy over a period of 14 days under repair conditions, led to no detectable misrejoining in wild-type cells but revealed a misrejoining frequency of 10% in NHEJ-deficient cells. This shows that in situations of separated breaks, NHEJ deficiency leads to genomic rearrangements, in agreement with chromosomal studies. However, if multiple DSBs coincide, even wild-type cells form genomic rearrangements frequently. These repair events are absent in Ku80-, DNA-PKcs-, and DNA ligase IV-deficient cells but are present in RAD54(-/-) cells. This strongly suggests that NHEJ has, in addition to its caretaker role, also the potential to effect genomic rearrangements. We propose that it serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space.

AB - Two major pathways for repairing DNA double-strand breaks (DSBs) have been identified in mammalian cells, nonhomologous end-joining (NHEJ) and homologous recombination (HR). Inactivation of NHEJ is known to lead to an elevated level of spontaneous and radiation-induced chromosomal rearrangements associated with an increased risk of tumorigenesis. This has raised the idea of a caretaker role for NHEJ. It is, however, not known whether NHEJ itself can also cause rearrangements. To investigate, on the DNA level, the influence of a defect in NHEJ on the formation of genomic rearrangements, we applied an assay based on Southern hybridization that allows the identification and quantification of incorrectly rejoined DSB ends produced by ionizing radiation. After 80 Gy of X-irradiation at a high dose rate (23 Gy/min), wild-type cells repaired 50% of the induced DSBs within 24 h by incorrect rejoining. This frequency of DSB misrejoining is considerably reduced in NHEJ-deficient cells. Low-dose-rate experiments, in which the cells were exposed to 80 Gy over a period of 14 days under repair conditions, led to no detectable misrejoining in wild-type cells but revealed a misrejoining frequency of 10% in NHEJ-deficient cells. This shows that in situations of separated breaks, NHEJ deficiency leads to genomic rearrangements, in agreement with chromosomal studies. However, if multiple DSBs coincide, even wild-type cells form genomic rearrangements frequently. These repair events are absent in Ku80-, DNA-PKcs-, and DNA ligase IV-deficient cells but are present in RAD54(-/-) cells. This strongly suggests that NHEJ has, in addition to its caretaker role, also the potential to effect genomic rearrangements. We propose that it serves as an efficient pathway for rejoining correct break ends in situations of separated breaks but generates genomic rearrangements if DSBs are close in time and space.

KW - Animals

KW - CHO Cells

KW - Cell Line

KW - Cricetinae

KW - DNA/genetics

KW - DNA Damage

KW - DNA Helicases

KW - DNA Ligase ATP

KW - DNA Ligases/metabolism

KW - DNA Repair/genetics

KW - DNA Repair Enzymes

KW - DNA-Activated Protein Kinase

KW - DNA-Binding Proteins

KW - Fibroblasts

KW - Fungal Proteins/genetics

KW - G1 Phase/physiology

KW - Gene Rearrangement/radiation effects

KW - Humans

KW - Mice

KW - Nuclear Proteins

KW - Protein-Serine-Threonine Kinases/metabolism

KW - Saccharomyces cerevisiae Proteins

M3 - SCORING: Journal article

C2 - 11358801

VL - 61

SP - 3886

EP - 3893

JO - CANCER RES

JF - CANCER RES

SN - 0008-5472

IS - 10

ER -