A new assay capturing chromosome fusions shows a protection trade-off at telomeres and NHEJ vulnerability to low-density ionizing radiation

Abstract

Chromosome fusions threaten genome integrity and promote cancer by engaging catastrophic mutational processes, namely chromosome breakage-fusion-bridge cycles and chromothripsis. Chromosome fusions are frequent in cells incurring telomere dysfunctions or those exposed to DNA breakage. Their occurrence and therefore their contribution to genome instability in unchallenged cells is unknown. To address this issue, we constructed a genetic assay able to capture and quantify rare chromosome fusions in budding yeast. This chromosome fusion capture (CFC) assay relies on the controlled inactivation of one centromere to rescue unstable dicentric chromosome fusions. It is sensitive enough to quantify the basal rate of end-to-end chromosome fusions occurring in wild-type cells. These fusions depend on canonical nonhomologous end joining (NHEJ). Our results show that chromosome end protection results from a trade-off at telomeres between positive effectors (Rif2, Sir4, telomerase) and a negative effector partially antagonizing them (Rif1). The CFC assay also captures NHEJ-dependent chromosome fusions induced by ionizing radiation. It provides evidence for chromosomal rearrangements stemming from a single photon-matter interaction.

Figure 1.

An assay to capture chromosome fusions. (A) CEN6 elimination stabilizes fusions between chromosome 6 and another chromosome. If chromosome 6 is unfused, CEN6 elimination generates an unstable acentric chromosome leading to cell death. (B) Schematic representation of the loxP cassette inserted at CEN6. (C) Survival to CEN6 loss of wild-type and NHEJ-deficient (lig4Δ) cells. Expo.: cells growing exponentially in rich medium (OD_600nm < 1). Stat.: cells reaching stationary phase in rich medium (3 days, OD_600nm ≈ 25). Each data point from an independent cell culture spread on a single plate. (D) Survival to CEN6 loss of wild-type cells reaching stationary phase from exponential growth (day 0, OD_600nm ≈ 2). (E) Karyotype of cen6Δ clones from wild-type stationary cells. In clones b and n, chromosome 6 is fused to chromosome 12.

Figure 2.

Telomere fusions in wild-type cells. (A) Telomere fusions at chromosome 6 telomeres among cen6Δ clones from wild-type stationary cells. Same clones as in Figure 1D. TEL6L and TEL6R positions marked with blue arrows. Cross-hybridizing restriction fragments marked with asterisks. (B) PCR amplification of fusions between chromosome 6 right telomere (TEL6R) and Y′-containing telomeres.

Figure 3.

NHEJ-dependent chromosome fusions in response to defects in telomere function. (A) Survival frequency to CEN6 loss in stationary cells lacking Rif2, Sir4, Rif1, Sir3, Tel1 and telomerase template RNA (TLC1). +EtOH: growth for 10 or 20 generations in glucose-containing rich medium complemented with 5% ethanol followed ∼5 generations in glucose-containing rich medium lacking ethanol to reach stationary phase. +Dox: growth to saturation for 10 or 20 generation in glucose-containing rich medium complemented with 30 μg/ml doxycycline. Telomere length in stationary phase at the time of plating shown in Supplementary Figure S1B. (B) Telomere fusions at chromosome 6 right telomere among cen6Δ clones from wild-type and mutant stationary cells.

Figure 4.

NHEJ-dependent chromosome fusions induced by ionizing radiation. (A) Survival to CEN6 loss in response to gamma-ray irradiation (¹³⁷Cs). (B) Cell survival in response to gamma-ray irradiation. (C) Karyotype of cen6Δ clones from irradiated wild-type stationary cells. (D) Fusion point sequence in cen6Δ clones from irradiated wild-type stationary cells. Blue: chromosome 6 sequence. Underlined: microhomology at the junction. Red: added non-homologous base. Distance from the closest telomere in kb.

Figure 5.

Impact of photon energy and dose fractionation on the frequency of radiation-induced rearrangements. (A) Survival to CEN6 loss in response to X-ray irradiations. (B) Survival to CEN6 loss in response to UVC irradiations. (C) Survival to CEN6 loss in response to dose fractionation; 24-h interval and incubation at 30°C between the two half-dose irradiations (¹³⁷Cs). (D) Dose fractionated four times; 24-h interval and incubation at 30°C between the quarter-dose irradiations (¹³⁷Cs).

Figure 6.

Impact of radiation density and telomere protection on the frequency of radiation-induced rearrangements. (A) Impact of the time between two irradiations of 5 Gy (¹³⁷Cs). (B) Impact of a lower dose rate (¹³⁷Cs). (C) Impact of a first irradiation on the response to a second irradiation; 24-h interval and incubation at 30°C between the two irradiations (¹³⁷Cs). (D) Impact of Rif2 and Rif1 loss (¹³⁷Cs).

Figure 7.

Model for NHEJ-dependent chromosomal rearrangements induced by single photon–matter interaction. Localized ionization and ROS formation lead to two closely spaced DSBs and consequently frequent inaccurate end synapsis and repair by NHEJ.