Clustered DNA damage induced by gamma radiation in human fibroblasts (HF19), hamster (V79-4) cells and plasmid DNA is revealed as Fpg and Nth sensitive sites

Abstract

The signature DNA lesion induced by ionizing radiation is clustered DNA damage. Gamma radiation-induced clustered DNA damage containing base lesions was investigated in plasmid DNA under cell mimetic conditions and in two cell lines, V79-4 (hamster) and HF19 (human), using bacterial endonucleases Nth (endonuclease III) and Fpg (formamidopyrimidine DNA glycosylase). Following irradiation with 60Co gamma-rays, induction of double-strand breaks (DSB) and clustered DNA damage, revealed as DSB by the proteins, was determined in plasmid using the plasmid-nicking assay and in cells by either conventional pulsed field gel electrophoresis or a hybridization assay, in which a 3 Mb restriction fragment of the X chromosome is used as a radioactive labeled probe. Enzyme concentrations (30-60 ng/microg DNA) were optimized to minimize visualization of background levels of endogenous DNA damage and DSB produced by non-specific cutting by Fpg and Nth in cellular DNA. 60Co gamma-radiation produces a 1.8-fold increase in the yields of both types of enzyme sensitive sites, visualized as DSB compared with that of prompt DSB in plasmid DNA. In mammalian cells, the increase in yields of clustered DNA damage containing either Fpg or Nth sensitive sites compared with that of prompt DSB is 1.4-2.0- and 1.8-fold, respectively. Therefore, clustered DNA damage is induced in cells by sparsely ionizing radiation and their yield is significantly greater than that of prompt DSB.

Figure 1

Dependence of the loss of closed circular plasmid DNA on γ- radiation dose (at 4°C) after subsequent treatment with buffer at 37°C in the absence (closed squares) and presence (open triangles) of Nth. This is an example of data from one experiment.

Figure 2

Dependence of increase in linear plasmid DNA on γ-radiation dose (at 4°C) after subsequent treatment with buffer at 37°C in the absence (open triangles) and presence (open squares) of Nth. This is an example of data from one experiment.

Figure 3

Dependence of the induction of prompt DSBs, presented as % FAR, in both HF19 (open circles) and V79-4 (closed circles) cells on dose of ⁶⁰Co γ-radiation. Lysis of cells in the FAR assay was performed with proteinase-K (see Materials and Methods). Error bars represent the SEMs calculated from at least three experiments.

Figure 4

Dependence of the induction of DSBs, presented as % FAR, on Fpg concentration in V79-4 cells either un-irradiated (closed triangles) or irradiated by 20 Gy ⁶⁰Co γ-radiation (closed squares). Lysis of cells in the FAR assay was performed with NaCl (see Materials and Methods). Error bars represent the SEMs calculated from at least three experiments.

Figure 5

Dependence of the induction of DSBs, presented as % FAR, on Fpg concentration in HF19 cells either un-irradiated (open triangles) or irradiated by 20 Gy ⁶⁰Co γ-radiation (open squares). Lysis of cells in the FAR assay was performed with proteinase-K (see Materials and Methods). Error bars represent the SEMs calculated from at least three experiments.

Figure 6

Dependence of the induction of DSBs on Nth concentration in HF19 cells either un-irradiated (open squares) or irradiated by 20 Gy ⁶⁰Co γ-radiation (open circles) and measured using the hybridization assay. Error bars represent the SEMs calculated from at least three experiments.