Processing of clustered DNA damage generates additional double-strand breaks in mammalian cells post-irradiation

Abstract

Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cells. Mutant hamster cells (xrs-5), deficient in non-homologous end joining (NHEJ), were irradiated at 37 degrees C to determine whether any additional double-strand breaks (DSBs) are formed during processing of gamma-radiation-induced DNA clustered damage sites. A class of non-DSB clustered DNA damage, corresponding to approximately 30% of the initial yield of DSBs, is converted into DSBs reflecting an artefact of preparation of genomic DNA for pulsed field gel electrophoresis. These clusters are removed within 4 min in both NHEJ-deficient and wild-type CHO cells. In xrs-5 cells, a proportion of non-DSB clustered DNA damage, representing approximately 10% of the total yield of non-DSB clustered DNA damage sites, are also converted into DSBs within approximately 30 min post-gamma but not post-alpha irradiation through cellular processing at 37 degrees C. That the majority of radiation-induced non-DSB clustered DNA damage sites are resistant to conversion into DSBs may be biologically significant at environmental levels of radiation exposure, as a non-DSB clustered damage site rather than a DSB, which only constitutes a minor proportion, is more likely to be induced in irradiated cells.

Figure 1

Overall rejoining of DSBs induced in (A) V79-4 wild-type hamster cells and (B) xrs-5 cells γ-irradiated at 37°C with a dose of 15 Gy delivered in 1 min and subsequently incubated at 37°C for up to 180 min. The yields of DSBs determined using the FAR assay are presented as percent breaks remaining after normalizing the initial yield to 100%.

Figure 2

Overall rejoining of DSB in V79-4 (open circles) and xrs-5 (filled circles) cells γ-irradiated with a dose of 15 Gy delivered in <1 min and subsequently incubated at 37°C for up to 30 min. The yields of DSB measured using the FAR assay are presented as percent breaks remaining after normalizing the initial yield to 100%.

Figure 3

Correct rejoining measured using the hybridization assay for wild-type hamster (A) and xrs-5 (B) cells irradiated at 4°C with a dose of 80 Gy of ⁶⁰Co γ-radiation. In (A) and (B) the pulsed field gel of irradiated, lysed, MluI-restricted DNA was stained with ethidium bromide (top) and blotted onto nylon membrane for hybridization to probe ATCC 77273 (bottom). S.P. and S.C. are S.pombe and S.cerevisiae chromosomes as size markers, respectively.

Figure 4

Percentage of mis- or un-rejoined DSBs measured using the hybridization assay in wild-type CHO-K1 (circles) and xrs-5 (triangles) cells irradiated at 4°C with a dose of 80 Gy of ⁶⁰Co γ-radiation and subsequently incubated at 37°C for various times up to 60 min. Yields are presented as percent mis- or un-rejoined DSBs remaining after normalizing the initially induced yields to 100%.

Figure 5

(A) Yield of DSBs expressed as percent FAR in V79-4 cells γ-irradiated with 15 Gy at different temperatures and (B) time for rejoining of DSBs induced by γ-irradiation of V79-4 cells with 18 Gy delivered within 0.5 min and at 37°C.

Figure 6

Overall rejoining of DSBs in V79-4 cells α-irradiated with a dose of 20 Gy delivered in <1 min at 37°C and subsequently incubated at 37°C for up to 10 min. The yields of DSBs measured using the FAR assay are presented as percent breaks remaining after normalizing the initial yield to 100%.