Radioresistance of GGG sequences to prompt strand break formation from direct-type radiation damage

Abstract

As humans, we are constantly exposed to ionizing radiation from natural, man-made and cosmic sources which can damage DNA, leading to deleterious effects including cancer incidence. In this work, we introduce a method to monitor strand breaks resulting from damage due to the direct effect of ionizing radiation and provide evidence for sequence-dependent effects leading to strand breaks. To analyze only DNA strand breaks caused by radiation damage due to the direct effect of ionizing radiation, we combined an established technique to generate dehydrated DNA samples with a technique to analyze single-strand breaks on short oligonucleotide sequences via denaturing gel electrophoresis. We find that direct damage primarily results in a reduced number of strand breaks in guanine triplet regions (GGG) when compared to isolated guanine (G) bases with identical flanking base context. In addition, we observe strand break behavior possibly indicative of protection of guanine bases when flanked by pyrimidines and sensitization of guanine to strand break when flanked by adenine (A) bases in both isolated G and GGG cases. These observations provide insight into the strand break behavior in GGG regions damaged via the direct effect of ionizing radiation. In addition, this could be indicative of DNA sequences that are naturally more susceptible to strand break due to the direct effect of ionizing radiation.

Keywords: DNA damage; Direct effect; Gel electrophoresis; Guanine; Ionizing radiation; Radiation chemistry; Radiosensitivity.

Figure 1

Visualization of site-specific DNA strand breaks due the direct effect of IR damage by X-rays. Image of bands produced through denaturing PAGE analysis of samples of single-G double stranded irradiated to the indicated absorbed dose. Lane 1 contains the unirradiated single-G oligonucleotide (ODN). Lanes 2–6 received doses of 15, 30, 60, 120 and 240 kGy, respectively. The four lanes on the right contain cleavage products produced by Maxam-Gilbert sequencing used to identify bands produced through radiolytic cleavage. Bands labeled with an arrow exhibit apparent precursor/product relationships, as a function of dose.

Figure 2

A plot showing the volume analysis of 3 example fragments of the single-G ODN at the indicated dose. Site-specific DNA strand breaks induced via the direct effect of IR increase with dose. For each fragment on both single- and triple-G ODN a linear regression was applied and slopes were calculated to provide a metric for strand break probability at that site. Parentheses around the final base indicated in the legend signify the base lost due to cleavage as a result of the irradiation. Dose response of radiation-generated fragments for the (B) single-G and (C) triple-G sequences. Relative cleavage probabilities as a function of dose were collected. Error bars represent one standard deviation.

Figure 3

Normalized dose response for radiation-generated fragments. Normalization of data shown in Figure 2 based on rolling average within each data set to emphasize relative trends. Both single- and triple-G substrates are represented. Error bars represent the standard deviation for each group of data.