Measuring radiation-induced DNA damage in Cryptococcus neoformans and Saccharomyces cerevisiae using long range quantitative PCR

Abstract

DNA damage has been considered to be the universal critical lesion in cells after exposure to ionizing radiation. Measuring radiation-induced DNA damage is important to understand the mechanisms of radiation-induced toxicity and monitor DNA damage repairs. Currently the most widely used methods to measure DNA damage are pulsed-field gel electrophoresis (PFGF) and single-cell gel electrophoresis (also known as the comet assay), both of which are technically challenging and time consuming. Long range quantitative polymerase chain reaction (LR-QPCR) has been used successfully to measure nuclear and mitochondrial DNA damage in mammalian and several model organism cells. The principle of this assay is that DNA lesions will slow down or block the progression of DNA polymerase. Therefore, the amplification efficiency of DNA with fewer lesions will be higher than DNA with more lesions under the same reaction condition. Here, we developed the LR-QPCR assay primers and reaction conditions to quantify DNA damage in Cryptococcus neoformans (C. neoformans) and Saccharomyces cerevisiae (S. cerevisiae) after gamma ray exposure. Under these conditions, long DNA targets of C. neoformans H99 and S. cerevisiae BY4741 (17.6 and 16.4 kb for nuclear DNA and 15.3 and 14.6 kb for mitochondrial DNA) were quantitatively amplified using extracted DNA templates, respectively. Two short mitochondrial DNA targets of these two species (207 bp and 154 bp) were also quantitatively amplified and used to monitor the number of mitochondria. Using the LR-QPCR method, we showed that the frequency of radiation-induced mitochondrial and nuclear DNA lesions had a significant linear correlation with the radiation doses (from 500 Gy to 3000 Gy) in both species. Furthermore, the faster disappearance of DNA damage detected in C. neoformans H99S strain compared to H99 strain may help to explain the different radiation sensitivity of these two strains. In summary, we developed a simple, sensitive method to measure radiation-induced DNA damage, which can greatly facilitate the study of radiation-induced toxicity and can be widely used as a dosimetry in radiation-induced cell damage.

Fig 1. Amplification of long mtDNA and nDNA and short mtDNA from C. neoformans H99 and S. cerevisiae BY4741 DNA extract.

(A and D) mitoLg, long mitochondrial DNA fragments from H99 and BY4741, (B and E) nLg, long nuclear DNA fragments from H99 and BY4741, (C and F) mitoSt, short mitochondrial DNA fragments from H99 and BY4741. M1, M2 and M3 are different size DNA ladders. FL, full length; Cut, restriction enzyme digested full length DNA.

Fig 2. Quantitative amplification of target DNA fragments in LR-QPCR with respect to the amount of template DNA.

DNA isolated from C. neoformans H99 and S. cerevisiae BY4741 were serially diluted with nuclease-free water for use in PCR. PCR of each sample was performed in triplicate for 26 cycles (long mitochondrial and nuclear DNA) and 20 cycles (short mitochondrial DNA), and the PCR products were quantified using the PicoGreen assay. (A and D) long mitochondrial DNA fragments from H99 and BY4741, (B and E) long nuclear DNA fragments from H99 and BY4741, (C and F) short mitochondrial DNA fragments from H99 and BY4741. The solid lines connect all of data points. The dotted lines are the linear regression lines. Results were from three independent experiments with triplicate samples in each experiment (3 samples/point/experiment x 3). Mean ± S.E.M.

Fig 3. Quantitative detection of DNA damage in C. neoformans H99 and S. cerevisiae BY4741 exposed to ionizing radiation using the long mitochondrial DNA and nuclear DNA fragments.

H99 and BY4741 cells were exposed to ionizing radiation from doses of 500 Gy to 3000 Gy. Irradiated cells were immediately frozen at -80^°C and DNA extraction was performed as described in the Material and Methods. PCR products were quantified using the PicoGreen assay. DNA lesions were calculated according to the formula (lesions/amplified fragment = -ln (A_D/A_C)). The solid lines are lines connecting each data point. The dotted lines are the linear regression lines. (A and D) long mitochondrial DNA fragment lesions, (B and E) long nuclear DNA fragment lesions, (C and F) short mitochondrial DNA fragment PCR yield. Results were from three independent experiments with triplicate samples in each experiment (3 samples/point/experiment x 3). Mean ± S.E.M. **, p<0.01; ***, p<0.001.

Fig 4. Different radio-sensitivity of C. neoformans H99S vs H99 strains and their DNA damage progression after 1 kGy exposure.

(A) H99S and H99 cells were cultured in YPD overnight, and then 10-fold serially diluted (10² to 10⁵) in PBS and 3 μL of diluted solution was spotted onto the YPD plates. Cells were exposed to the indicated doses of γ-radiation and then further incubated at 30^°C for 5 days. Image was from one representative experiment of three independent experiments. (B) & (C) DNA damage progression of C. neoformans H99S and H99 cells after 1 kGy γ-radiation measured by mitochondrial DNA and nuclear DNA LR-QPCR, respectively. After radiation, cells were harvested at selected time points and DNA was extracted and PCR was performed using 1 ng DNA and 30 ng DNA for the long mitochondrial DNA fragment and long nuclear DNA fragment respectively. Results were from three independent experiments with triplicate samples in each experiment (3 samples/point/experiment x 3). Mean ± S.E.M. **, p<0.01; ***, p<0.001.