Mutagenesis Advance Access published online on February 10, 2008
Mutagenesis, doi:10.1093/mutage/gem055
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Involvement of homologous recombination repair after proton-induced DNA damage
Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, B313 Health Sciences Building, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada 1Department of Radiation Medicine, Loma Linda University, Loma Linda, CA 92324, USA 2Department of Microbiology and Immunology, College of Medicine, University of Saskatchewan, B313 Health Sciences Building, 107 Wiggins Road, Saskatoon, SK S7N 5E5, Canada
Protection from chronic exposure to cosmic radiation, which is primarily composed of protons, in future manned missions to Mars and beyond is considered to be a key unresolved issue. To model the effects of cosmic radiation on a living cell, we used Saccharomyces cerevisiae cells harboring various deletions of DNA repair genes to investigate the response of cells to DNA strand breaks caused by exposure to 250 MeV proton irradiation (linear energy transfer of 0.41 keV/µm). In our study, DNA strand breaks induced by exposure to protons were predominantly repaired via the homologous recombination and postreplication repair pathways. We simulated chronic exposure to proton irradiation by treating cells from colonies that survived proton treatment, after several rounds of subculturing, to a second proton dose, as well as additional cell stressors. In general, cells cultured from proton surviving colonies were not more sensitive to secondary cell stressors. However, cells from rad52
colonies that survived proton treatment showed increased resistance to secondary stressors, such as 
-rays (1.17 and 1.33 MeV; 0.267 keV/µm), ultraviolet (UV) and proton irradiation and elevated temperatures. Resistance to secondary stressors was also observed in rad52 cells that survived exposure to -rays, rather than protons, but this was not observed to occur in rad52 cells after UV irradiation. rad52 cells that survived exposure to protons, followed by -rays (proton surviving colonies were cultured prior to -ray exposure), exhibited an additive effect, whereby these cells had a further increase in stress resistance. A genetic analysis indicated that increased stress resistance is most likely due to a second-site mutation that suppresses the rad52 phenotype. We will discuss possible origins of these second-site mutations.
 |
Introduction |
|---|
 Top Introduction Materials and methodsResultsDiscussionFundingReferences |
|---|
Although the prospect of manned missions to Mars and beyond is becoming more than mere science fiction (1
–3), numerous barriers must be overcome, ranging from space flight technology to human biology. One of the primary concerns for human space travel is chronic exposure to damaging cosmic radiation (4–7). While estimates and measurements of cosmic radiation have been reported (including those obtained from the space station), the effects of chronic long-term exposure have yet to be thoroughly studied (8,9). It has been estimated that humans can withstand 
1 mSv per day, which is almost 150 times higher than cosmic radiation on the surface of the Earth (10). Previous estimates on radiation exposure have suggested that a Mars mission could potentially yield an accumulated exposure higher than 1 mSv per day during the course of a 3-year mission, and a solar flare event would potentially increase this dose (11). However, the long-term impact of cosmic radiation on the health of astronauts traveling to Mars remains unknown.
To model the effects of cosmic radiation on a living cell, we chose to pursue land-based experiments utilizing the proton accelerator at Loma Linda University Medical Center (LLUMC). Cosmic radiation is primarily composed of protons (95%) with other ions (Fe, C, etc.) completing the radiation profile (12). A large proportion of cosmic radiation in low-Earth orbit, where astronauts are spending extended periods of time today, are composed of protons [(13) and references within]. Typically, astronauts are exposed to combined energies of 40 MeV. However, in some regions of low-Earth orbit, such as in the southern hemisphere, proton energies can reach between 50 and 200 MeV. Thus, it is imperative to understand the effects on a biological specimen chronically exposed to such conditions. For ease of study and genetic manipulation, we characterized the effects of proton irradiation on Saccharomyces cerevisiae cells expressing a range of mutations in genes involved in DNA repair. Based on the striking conservation of DNA repair proteins from yeast to humans, data gained from studying model systems, such as yeast, can be directly applicable to studies of higher eukaryotic systems (14,15).
Protons, as well as other particles and photons, induce DNA strand breaks (16,17). In addition to strand breaks, abasic and oxidized base damage occurs. The combination of these events can result in complex bistrand clusters. Single-strand breaks (SSBs), as well as abasic and oxidized base damage, on opposite strands within several helical turns can cause DNA double-strand breaks (DSBs). These clusters tend to be refractory to repair, and can be converted to DSBs during attempted repair. It has been suggested that the decrease in DSBs with increasing linear energy transfer (LET) reflects the accumulation of clusters where small segments of the genome suffer multiple lesions (18,19). This is supported by observations that protons and alpha particles produce a significant proportion of small DNA fragments that cannot be explained by randomness (20). These small DNA fragments are believed to increase with increasing LET (20,21). The presence of clusters also suggests a possible explanation for why there is little increase in DSBs with increasing ionization but a large increase in the relative biological effectiveness [RBE; (22,23)]. A comparison of different ionizing radiations showed that charged particles produced a greater number of DSBs compared to abasic and oxidized base clusters than ionizing photons, with protons generating the highest ratio of DSBs to abasic and oxidized damage (18).
The induction of DNA damage by low-energy particles in human cells was demonstrated by using immunocytochemistry to image the accumulation of DNA damage inducible proteins and histone H2AX along ion trajectories (24,25). mRNAs responding to damage induced by proton irradiation include those that encode Rad51, ATM, p73, p21, SOD, Bcl2 and Bax
(26,27). Rad51 and ATM are recruited to sites of damage in human cells (19). The induction of SOD was consistent with an increase in intracellular free radicals in response to protons (27). Other agents can induce DSBs, such as methyl methanesulfonate [MMS; (28)]. MMS methylates DNA to produce 3-methyladenine (29), which is mainly repaired by the base excision repair (BER) pathway. It is believed that 3-methyladenine blocks replication, which, if not repaired correctly by BER, will cause DSBs (30–32). Another source of irradiation that is solar in origin is ultraviolet (UV) irradiation. UV induces cyclobutane pyrimidine dimers and (6–4) photoproducts that are mainly corrected by nucleotide excision repair (NER). However, DSBs can result when replication forks collide with UV-induced cyclobutane pyrimidine dimers (33). Yeast cells express a broad array of proteins that repair photon-induced damage, including BER, NER and DNA postreplication repair (PRR) that can be error prone or error free (34), homologous recombination [HR; (35)] and nonhomologous end joining [NHEJ; (36)]. NHEJ in yeast is most efficient on endonuclease-induced DSBs, whereas DSBs produced by ionizing radiation make poor substrates (37). Further characterization of the repair pathways responsible for the repair of proton-induced DNA damage will provide valuable insight in terms of surviving chronic exposure to cosmic radiation.
Different radiation sources that induce strand breaks can have different outcomes in regards to cluster complexity and RBE. For example, damage induced by treatment of cells with protons appears to be more serious than damage induced by photon irradiation (27,38,39). Ianzini et al. (38) showed that both protons and X-rays induce mitotic catastrophe in Chinese hamster V79 cells, but protons are more effective. Likewise, Antoccia et al. (39) and Di Pietro et al. (27) showed that a variety of human cells treated with protons or X-rays had subtle differences. Both treatments resulted in G2 arrest. However, Antoccia et al. (39) reported that G2 arrest and p21 induction was more prominent with protons, and Di Pietro et al. (27) reported that apoptosis was more prominent using protons compared to X-rays. Several studies have now shown that protons produce smaller DNA fragments than - and X-rays that cannot be explained by randomness, suggesting that protons produce more complex DNA lesions than - or X-rays (18,20,40,41). In fact, it has been suggested that low-LET protons would have a greater impact on biological systems than photons when compared to high-LET particles (18). In this study, we describe the results of our investigations into the effect of low-LET proton irradiation at the molecular level in S.cerevisiae.
|
Materials and methods |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Yeast strains and methods
The yeast strains used in this study and their sources are listed in Table I. The sequence between the HpaII site immediately upstream of the RAD52 start site and the SphI site 15 base pairs upstream of the RAD52 stop were replaced with the LEU2 gene (42
). All other mutations were previously reported as null alleles. The asynchronous cultures used for stress exposure experiments were in early to late log-phase growth. Typically, the relative survival differences inherent to the various strains in the presence and absence of treatment were measured by spot dilutions using the appropriate media. Spot dilutions were conducted by setting up overnight cultures in 2 ml of rich YPD medium (1% yeast extract, 2% peptone and 2% glucose). The next day, spectroscopic optical density readings were measured at a wavelength of 600 nm (OD600; OD600 of 1.0 
2 x 107 cells/ml) to determine cell concentration. Yeast cultures were then set up in 1 ml YPD at a concentration of 2 x 107 cells/ml. Tenfold serial dilutions were prepared and 5 µl of each dilution was spotted onto plates. The plates were then treated according to the following: (i) for MMS treatments, YPD plates were supplemented with 0.005, 0.01 and 0.025% MMS after autoclaving and (ii) for UV treatments, cells were spot diluted and exposed to 254-nm UV light in a UV cross-linker (Fisher Science model FB-UVXL-1000 at 2400 µW/cm2) at given doses in the dark. The plates were wrapped in foil and incubated at 30°C for 3 days. Cells were exposed to -rays using a 60Co -ray source (Department of Chemistry, University of Saskatchewan) at a dose rate of 893.55 rad/min [8.94 gray (Gy)/min]. Two -rays were emitted from the 60Co -ray source, at energies of 1.17 and 1.33 MeV. Energies of 1.17 and 1.33 MeV from a 60Co -ray source were previously reported to have a LET of 0.267 keV/µm (43). UV spot dilutions were repeated at least three times, while the -ray and MMS spot dilutions were conducted twice. Plates were scanned using an Epson Perfection 1650. Genetic analyses were performed as previously described (44). Four full tetrads from rad52 control crosses and five from the 200-Gy proton survivor crosses (YTH3223) were picked. The data shown are representative of the data obtained.
|
Proton irradiation
The proton accelerator at LLUMC was the source of protons utilized in this study. The experimental arrangement of the proton accelerator used was described previously (13
). Control plates were manipulated similar to irradiated plates. The dose rate was 0.6 Gy/min at an energy of 250 MeV (237 MeV at target with a LET of 0.41 keV/µm). LET values at LLUMC were previously reported as 0.39 keV/µm for an energy of 249 MeV (43) and 0.5 keV/µm for an energy of 172 MeV, in which a 38-mm polycarbonate absorber was used (13). Calibration and charge readings were performed by placing an ion chamber (PTW Markus parallel plate) at the target. This was performed and calculated at least three times or until the readings agreed. These readings were then compared to a detector upstream, which detected the number of counts upstream that equal 1 Gy at the target. After calculation, the ion chamber was removed and the plates were placed at the target. The region before the entrance of the Bragg curve was used. The peak of the Bragg curve was monoenergetic, meaning there was no range shifting and the peak was not spread out. Yeast plates were exposed to protons in at least three separate experiments with reproducible results. It was not necessary to grow cells in the dark following exposure to protons and -rays as photoreactivation does not repair strand breaks.
Survival curves
Overnight cultures (2 ml) of yeast strains were grown at 30°C in YPD liquid. Cell concentrations were determined by OD600 measurements. Serial dilutions were performed and a known number of cells (50–100 cells/plate) were plated on YPD plates containing 2% agar. Cells were exposed to 254-nm UV light in a UV cross-linker at the doses indicated. The plates were wrapped in foil along with the unexposed control plates and incubated for 3 days at 30°C prior to counting. Proton irradiation survival curves were performed in the same manner. The same number of cells were plated on a series of plates and placed under the proton beam. After a certain dose was achieved, the appropriate plates were removed. The colonies on all plates were then manually counted and the number of surviving colonies on exposed plates was compared to the number of colonies present on unexposed plates (of the same number of cells plated) to determine survival percentage. Most experiments were done at least three times in duplicate. The curve presented in Figure 1B was typical of the results obtained from that experiment.
|
Multiple exposure protocol
Treatment of cells with more than one stress was conducted as follows. Individual colonies that survived the primary proton irradiation were selected, cultured and stored as permanent glycerol stocks at –80°C. These cells were then repropagated on YPD and exposed to UV,
-rays, elevated temperatures or protons as described above. Again, individual colonies that survived these treatments were selected, cultured and stored at –80°C.
Statistical analysis
Results were graphed and error bars were determined using standard error of the mean. Generally, mean ± standard error (for the number of experiments designated) was reported. In some cases, the error bars were smaller than the symbol representing the curve.
|
Results |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Yeast cells lacking HR and PRR repair pathways are sensitive to proton irradiation
We employed S.cerevisiae in our analysis of the DNA repair mechanisms used to repair damage arising from proton irradiation. Yeast cells harboring gene deletions for specific repair enzymes involved in NER (rad1
), PRR (rad18), HR (rad52), BER (apn1 apn2) and mitotic checkpoints (mec1) were spot diluted onto YPD plates and exposed to increasing doses of protons. Isogenic wild-type strains for apn1 apn2, rad1, rad18 and rad52 and for mec1 were used (generously provided by D. Botstein and A. Emili, respectively). As shown in Figure 1A, wild-type yeast cells exposed to 150 Gy grew slower than untreated controls. We found that wild-type cells continued to grow, albeit at a much slower rate, when treated with as much as 200 Gy (data not shown). Our results are consistent with previous findings demonstrating that wild-type yeast cells can withstand high doses of low-LET ionizing radiation [200–500 Gy, using a Varian Linear Accelerator with a 6-MeV electron beam at 38 Gy/min.; (45)]. Cells lacking RAD1 or MEC1, or both APN1 and APN2, showed modest decreases in survival. As expected for a strand break-inducing agent such as protons, cells lacking RAD18 or RAD52 were highly sensitive to proton irradiation (Figure 1A). This is consistent with early reports demonstrating that PRR and HR mutants, but not NER mutants, are sensitive to DNA strand break-inducing agents such as - and X-rays and the radiomimetic bleomycin (46–52). Indeed, HR mutants were originally isolated by their sensitivity to ionizing irradiation [reviewed in (53)]. As both Rad52p and Rad18p, belonging to HR and PRR, respectively, are involved in handling strand breaks, among other types of DNA damage, our results support previous studies showing that the primary lesions in yeast induced by proton irradiation are strand breaks (17,54).
Proton survival curves were determined for the mutants shown in Figure 1A. The curves shown in Figure 1B were typical of the results obtained. The two wild types, DBY747 (isogenic to rad1, rad18 and rad52) and YMP10650 (isogenic to mec1), had identical survival curves. The survival curves support the conclusion drawn from the spot dilution approach (Figure 1A) as both mec1 and rad1 had modestly reduced survival curves, and rad18 and rad52 curves were more severely impacted. Thus, spot dilutions and survival curves demonstrate, as expected, that rad18 and rad52 cells are highly sensitive to proton irradiation.
To determine whether other mutants involved in HR were sensitive to proton irradiation, we assayed additional members of the RAD52 epistasis group for sensitivity to proton irradiation using the spot dilution method. We found that all tested members of this group (strains kindly provided by L. Symington) were sensitive to 150-Gy proton irradiation (Figure 1C). Thus, HR is a primary pathway responsible for repair of strand breaks induced by proton irradiation.
Cells from rad52 colonies that survive proton irradiation exhibit increased resistance to secondary stressors
As a simple means to mimic in yeast the effects of prolonged exposure to cosmic radiation, we asked whether proton irradiation survivors behaved differently from unexposed parental cells upon treatment with additional DNA damaging agents. We observed that cells from rad52 colonies that survived high doses of proton irradiation, when selected, cultured and exposed to protons for a second time, had a better survival curve (rad52 + 150 Gy and rad52 + 200 Gy reflect cells from individual colonies that survived these doses; Figure 1B). Growth of these colonies indicated that the original cell exposed to protons had repaired any DNA damage to a point that was compatible with life. To determine the generality of this observation, individual WT, apn1 apn2, mec1, rad1 and rad18 colonies that grew after exposure to 100-Gy protons were selected, cultured and exposed to additional stressors, including - and UV irradiation, MMS and elevated temperatures. Surviving exposure to a dose of 100-Gy protons had no effect on the susceptibility of these cells to UV exposure (Figure 2A). Furthermore, the selected rad1 and rad18 proton survivors responded to -rays, MMS and elevated temperatures similar to controls (Figure 2B and data not shown). UV survival curves (Figure 2C) showed that increased capacity to withstand a second stressor was only observed with the selected rad18 cells (marked by an ‘*’). Taken together, our results suggest that the yield of cells exhibiting enhanced stress resistance following proton irradiation is increased using cells defective for DNA strand break repair.
|
We continued our analysis of rad52
cells that grew after exposure to proton irradiation by evaluating the response of these cells to a variety of stresses. We observed that cells cultured from individual rad52 colonies that survived increasing doses of proton irradiation had a dose-dependent increase in resistance to elevated temperatures and UV radiation (Figure 3A and B). UV survival curves also displayed a dose-dependent increase in stress resistance following proton irradiation (Figure 3C). The effect was most apparent with rad52 cells obtained from individual colonies that grew following exposure to 200-Gy protons as these cells behaved similar to wild type in our experiments (Figure 3A and C).
|
We next tested additional HR mutants for enhanced resistance to UV following proton irradiation (Figure 4). Two colonies of rad50
, rad51, rad57 and xrs2 cells that grew following exposure to 150 Gy of proton irradiation were selected, cultured and exposed to UV. A spot dilution analysis indicated very little change in sensitivity to increasing doses of UV following proton treatment (Figure 4A). UV survival curves also showed little change compared to untreated cells (Figure 4B). Subtle differences were observed, however. For example, cells from one proton surviving rad50 colony showed increased sensitivity to UV, while one rad51 colony had a slight resistance to UV. The changes were not as dramatic as that observed for rad18 cells (Figure 2C) or rad52 cells (Figure 3). We also found little difference in the ability of proton-treated survivors to withstand -rays or enhanced temperatures (data not shown). Thus, cells lacking RAD52 are the most sensitive to this phenomena of HR mutants tested in this study.
|
Cells from rad52
colonies that survive -rays also exhibit increased stress resistanceTo determine if the selection of rad52
cells with increased stress resistance is a specific response to proton irradiation, we questioned whether UV irradiation or -rays were also capable of impacting the stress response. Cells from three rad52 colonies that survived UV irradiation (100 J/m2) and from four colonies that survived -rays (200 Gy) were selected, cultured and assayed for growth at 37°C (Figure 5A, panels ii and iii). The results show that cells from one of the three UV surviving colonies had a slight increase in growth at 37°C (compare panel i, row 2 with panel ii, rows 1–3), whereas cells from all four -irradiated survivors grew better at 37°C than the untreated control cells (compare panel i, row 2 with panel iii, rows 1–4). According to previous reports, protons generate more DSBs than -rays (18), and while UV produces mostly cyclobutane–pyrimidine dimers, DSBs can be produced when replication forks collide with UV-induced lesions (33). Thus, our data are consistent with the idea that the more potent the agent at generating DSBs, the greater the chance of selecting rad52 surviving cells with increased stress resistance.
|
Next, we asked if stress resistance in cells obtained from a single rad52
colony that survived 200-Gy protons could be further increased by a second exposure to stress (Figure 5A, panels iv and v). The rad52 cells utilized in this experiment (YTH3225) were obtained from a different 200-Gy proton surviving colony than the rad52 survivor used in Figures 1 and 3 (YTH3223). YTH3223 (used in Figures 1 and 3) grew better than parental rad52 cells at elevated temperatures and when exposed to UV. On the other hand, YTH3225 cells (rad52 + 200 Gy protons in Figure 5A), exhibited increased growth at elevated temperatures (Figure 5A, compare panel iv, row 1 with panel i, row 2), but were not resistant to UV (Figure 5D). The different phenotypes expressed in YTH3223 and YTH3225 could reflect the selection of different rad52 suppressing mutations in the different colonies. Nonetheless, this phenotypic difference allowed us to observe additional affects of -rays on the rad52 proton survivor phenotype. Comparing the growth of cells from parental rad52 colonies that survived UV and (Figure 5A, panels ii and iii) with cells from rad52 + 200 Gy proton colonies at 37°C (Figure 5A, panel iv, row 1) indicated that cells from colonies that survived -rays, but not cells from UV surviving colonies, were as equally effective at growth at the restrictive temperature as those that survived protons. Furthermore, growth at 37°C demonstrated that cells from rad52 + 200 Gy proton colonies that grew following subsequent UV irradiation (Figure 5A, panel iv, rows 2 and 3) was similar to the control cells (Figure 5A, panel iv, row 1), suggesting that UV exposure had little impact on the stress resistance of these cells. On the other hand, exposure of rad52 + 200 Gy proton cells to a subsequent dose of -rays (200 Gy) had a much greater impact on the cell's ability to deal with additional stressors (Figure 5A, panel v). Cells from two separate rad52 + 200 Gy proton colonies that grew following -ray treatment were selected, cultured and then spot diluted for growth at elevated temperatures. While one survivor lost all benefits first acquired after treatment with protons (Figure 5A, panel v, row 1), the second survivor had a capacity to grow at 37°C that was similar to wild type (Figure 5A, compare panel v, row 2 with panel i, row 1). Thus, an additive affect is possible when rad52 are exposed to multiple strand break-promoting agents in succession. It should be noted that DNA repair following proton exposure would be complete before exposure to -rays as the cells were cultured between treatments.
Similar experiments testing the ability of UV (100 J/m2) or -rays (200 Gy) to enhance the stress response of rad52 cells were conducted using UV survival curves. The rad52 and rad52 + 200 Gy proton cells that survived -rays and UV irradiation, as described in Figure 5A, were used in this experiment. Two biological replicates were performed, with two technical replicates each, generating four data sets for each sample. UV survival curves using rad52 parental cells from individual colonies that survived 100 J/m2 UV or 200-Gy -rays were similar to untreated rad52 cells with some moderate improvements (Figure 5B and C). UV survival curves of rad52 + 200 Gy proton survivors exposed to 100 J/m2 UV or 200-Gy -rays were then determined (Figure 5D and E). Treating rad52 + 200 Gy proton survivors with 100 J/m2 UV had no effect on the subsequent UV survival curve (Figure 5D). Exposure of rad52 + 200 Gy proton survivors to 200-Gy -rays produced a survivor with dramatically increased stress resistance (indicated by an ‘*’; Figure 5E). Thus, we repeatedly observed an increased probability of selecting rad52 cells that survived exposure to agents that induce strand breaks, such as -rays and proton irradiation, with increased resistance to subsequent secondary stressors. Furthermore, a regimen where rad52 proton surviving cells were selected, cultured and then submitted to a subsequent dose of -rays resulted in survivors with an additive effect; cells with stress resistance greater than single treatment survivors alone could be isolated.
Genetic nature of increased stress resistance in rad52 cells
To determine the genetic nature of increased stress resistance in rad52 cells, we crossed rad52 200-Gy proton survivors (YTH3223; the UV-resistant strain used in Figures 1 and 3) with a laboratory wild-type strain (YTH3). Use of YTH3223 allowed us to observe loss of enhanced UV irradiation resistance in the progeny. As a control, we used parental rad52 cells in crosses. All spores, from four control and five experimental tetrads analyzed, were checked for the presence of the LEU2 gene, which replaced the RAD52 gene in rad52 cells (WXY9387). With parental rad52 cells, we expected to generate rad52 spores that retained the parental UV survival curve profile. With rad52 + 200 Gy proton cells, if increased stress resistance was due to the selection of a rad52 cell harboring a second-site mutation, then we expected to generate rad52 spores that continued to express increased stress resistance and those that lost it due to segregation of the second-site mutation. Representative UV survival curves generated from a tetrad from the control cross showed that the two WT spores had the same survival curve as a wild-type parental strain, whereas rad52 spores were identical to the rad52 parental strain (Figure 6A). Figures 6B–D show representative results from three separate tetrads obtained from crosses involving rad52 + 200 Gy proton cells (YTH3223). In Figure 6B, both rad52 spores continued to exhibit increased resistance to UV irradiation, indicating either the phenotype is not due to a second-site mutation or that the two mutations did not segregate in this tetrad. In Figures 6C and D, one of the rad52 spores from each cross (marked by an ‘*’) generated a survival curve similar to the parental rad52 strain. These observations suggest that the original rad52 phenotype could be restored in rad52 + 200 Gy proton survivors through the segregation of a second-site mutation away from the rad52 allele. Thus, our data support the notion that a second-site mutation selected in response to surviving proton or -irradiation of a similar LET value can suppress rad52-heightened sensitivity to DNA damaging agents. Cloning of the second-site mutation in YTH3223 has so far been unsuccessful as this mutation does not produce an obvious phenotype. Future work will focus on identifying and characterizing second-site mutations in other rad52 and rad18 proton survivors with increased stress resistance. Identification of these mutations could yield insight into mechanisms of dealing with chronic exposure to cosmic irradiation and provide clues to ensuring resistance.
|
|
Discussion |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
In this report, we demonstrate the following findings: (i) damage induced by proton irradiation is repaired by the HR and PRR pathways, (ii) there is a higher probability of selecting rad52
cells with increased resistance to stress after exposure to protons or -rays of similar LET than with UV and (iii) this phenotype is likely conferred by second-site suppressor mutations. The results reported here form the framework for future studies focused on understanding the effects of chronic exposure to cosmic radiation on human space travelers.
In the present study, cells lacking proteins involved in BER (apn1 apn2) and NER (rad1) were weakly impaired in their response to protons (Figure 1A and B). Cells lacking MEC1 were also modestly sensitive to proton irradiation, indicating a need for checkpoints in responding to proton irradiation. However, cells lacking the PRR protein Rad18p or members of the RAD52 epistasis group, which defines the HR pathway, were severely impaired in response to proton irradiation (Figure 1). HR, considered error free, repairs mainly DSBs, while PRR, considered error prone, responds to any agent that causes replication blocks. PRR does not remove the lesion but enables the polymerase to synthesize past the single-strand gap (34). It has been shown that the PRR and HR pathways cooperate to maintain chromosomal DNA structure (34,55). Yeast predominantly relies on HR to repair DSBs and uses NHEJ as a backup. Opposed to the yeast system, NHEJ is predominantly used in mammalian systems (56). However, in yeast, damaged DNA ends generated by ionizing irradiation are poor substrates for the NHEJ machinery (37).
This study utilized a high energy, low-LET proton beam at LLUMC. Low-LET proton beams have been shown to produce predominantly single-track strand breaks that can be repaired by existing repair mechanisms (19,22). As the LET of a proton beam increases, the strand break frequency of both SSBs and DSBs increases and then abruptly decreases at higher LET values (18,19,21,43). This is believed to be due to the increasing complexity of the lesions induced. For example, as LET increases, complex clusters, in which abasic and oxidized DNA damage, and strand breaks occur within one or two helical turns of the DNA backbone. These clusters may appear experimentally as a single DSB. Hence, although DSB frequency does not increase at higher LET, RBE does since these complex clusters are generally refractory to repair. Thus, the use of low-LET protons and -rays in our experiments is predicted to result predominantly in well-spaced strand breaks that would, in general, be rapidly repaired in wild-type yeast cells. This is supported by the ability of wild-type yeast to withstand high doses (200–500 Gy) of low-LET protons (our work) and low-LET electrons (45). In our work, exposure to protons (LET of 0.41) had a greater impact on cell survival when compared to -rays (LET of 0.267). For example, rad18 cells had a better chance of surviving 200-Gy -rays than 150-Gy protons, and rad1 cells responded similar to wild type when exposed to 300-Gy -rays but had a decreased survival rate compared to wild type when exposed to 150-Gy protons (compare Figures 1A with 2B). This is consistent with previous studies that suggest protons produce more complex DNA lesions than - or X-rays (18,20,40,41). Our results show that yeast cells specifically mutated in HR and PRR pathways are compromised in their ability to respond to low-LET proton-induced damage. Yeast cells defective in NER and BER would have difficulty responding to abasic and oxidized DNA damage. Since this was not observed, our data are consistent with low-LET protons producing mainly strand breaks.
We demonstrate the selection of rad18 and rad52 proton irradiation surviving colonies that exhibited increased resistance to secondary stressors (Figures 1C, 2C and 3). A comprehensive frequency analysis was not performed in this study to determine the percentage of surviving colonies that harbored cells exhibiting increased stress resistance. Furthermore, our experiments cannot distinguish between whether this phenotype is due to selection of a previously existing mutation or the induction of a new mutation as a result of misrepair of DSBs. Moreover, we did not observe increased resistance to secondary stressors with other members of the RAD52 epistasis group (Figure 4). This may be due to the presence of Rad52p, which plays a vital step in initiating strand invasion (57). In fact, in the absence of RAD51, RAD54, RAD55 or RAD57, some types of recombinational repair can still occur. This may be due to the dependence of Rad52p, but not Rad51p, Rad55p or Rad57p, for the formation of Holliday junctions (58). Furthermore, the interplay between PRR and HR may allow efficient repair of strand breaks when components of HR other than Rad52p are mutated (34). Earlier work showed that mutagenic repair occurred in the absence of RAD50–57 (59) and that rad52 and rad18 mutants were spontaneous mutators (60–62), providing insight into a mechanism whereby suppressor mutations could be generated from exposure to DSB-inducing agents. The spontaneous mutator phenotype in rad52 mutants relied entirely on the translesion synthesis polymerase Rev3p (62). However, further work must be done to elaborate on this hypothesis as rad51 cells were also shown to be mutators (61,63), but we did not observe rad51 proton irradiation survivors with increased stress resistance in our limited study.
To test whether increased stress resistance in rad52 cells was specific to proton irradiation, we asked whether rad52 cells that survived UV or -rays would also exhibit increased stress resistance. Our results show that selection of stress resistant rad52 cells has a greater chance of occurring when agents that induce DNA strand breaks are used. We observed that rad52 cells exposed to -rays produced more survivors with increased stress resistance than with rad52 cells surviving UV irradiation (Figure 5A, and compare Figure 5B with C). Furthermore, exposure of rad52 200-Gy proton survivors to -rays, but not UV, resulted in an isolate that had attained stress resistance beyond what was observed in the survivor strain prior to exposure to -rays (Figure 5A, and compare Figure 5D with E). UV-induced lesions can be repaired by photoreactivation and NER in rad52 cells, which are error-free modes of repair. Since the bulk of UV-induced DNA damage repair is error free, our observation that cells from rad52 colonies that survive UV irradiation do not have increased stress resistance suggests that misrepair of strand breaks in rad52 cells that grew after ionizing irradiation may lead to the generation of new mutations that suppress rad52 phenotypes. Future studies will be aimed at determining whether the second-site suppressing mutations in rad52 cells are new or preexisting. Incorporating the rev3 and pol32 mutations into our studies, both of which abolish or reduce the mutator effect in rad52 cells, respectively (62,63), should assist in these studies.
It remained possible that increased stress resistance in rad18 and rad52 cells is a consequence of complex genetic interactions, simple gene mutations or contaminating background cells. Suppressor mutations of rad18 have been previously isolated and characterized, which include inactivation of the Srs2p helicase activity (64) and the Siz1p SUMO ligase (65). Mutations to genes of this nature in rad18 cells could result in increased resistance to secondary stresses in our experiments. In contrast, little is known regarding mutations capable of suppressing rad52 cells from killing by DNA damaging agents. The two rad52 + 200 Gy proton survivors utilized in this study, YTH3223 and YTH3225, responded differently to UV irradiation. This could be the result of different second-site mutations within these strains. To address whether increased stress resistance in rad52 cells is due to a second-site suppressor mutation, we crossed cells from a rad52 proton survivor that exhibited increased resistance to UV with a wild-type laboratory strain. Our data in Figures 6C and D demonstrate that we were indeed able to recover rad52 proton survivors that expressed parental sensitivity to UV irradiation after crosses with wild-type cells. This supports the notion that the increased stress resistance observed in rad52 proton survivors is due to a second-site mutation. What is this mutation? Unfortunately, the isolated second-site mutation did not have an obvious phenotype, rendering further characterization at this point difficult. Future work will focus on the characterization of additional rad18 and rad52 survivors. We expect to recover novel suppressive mutations within different rad52 proton surviving colonies.
The data presented here extend our knowledge of how DNA damage, whether acute or chronic, is processed in a living cell. Although it is unreasonable to select astronauts on the sole basis of whether their HR pathway is functioning efficiently, therapies that increase the activity of the HR pathway in humans may offer protection against ionizing radiation.
|
Funding |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
Canadian Foundation for Innovation (T.A.A.H.); a NASA Cooperative Agreement (NCC9-149 to A.O.).
|
Acknowledgments |
|---|
We thank D. Botstein, A. Emili and L. Symington for generously providing strains. The authors thank Dr G. Nelson for helpful discussions. Conflict of interest statement: None declared.
|
Notes |
|---|
* To whom correspondence should be addressed. Tel: 306 966 1995; Fax: 306 966 4298; Email: troy.harkness{at}usask.ca.
|
References |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionFundingReferences |
|---|
-
1. DeAngelis G, Clowdsley MS, Nealy JE, Tripathi RK, Wilson JW. Irradiation analysis for manned missions to the Jupiter system. Adv. Space Res. (2004) 34:1392–1403.
2. Chassefiere E, Bertaux JL, Berthelier JJ, Cabane M, Ciarletti V, Durry G, Forget F, Hamelin M, Leblanc F, Rochette P. MEP (Mars Environment Package): toward a package for studying environmental conditions at the surface of Mars from future lander/rover missions. Adv. Space Res. (2004) 34:1702–1709.[CrossRef][Web of Science][Medline]
3. Petrov VM. Problems and conception of ensuring irradiation safety during Mars missions. Adv. Space Res. (2004) 34:1451–1454.[CrossRef][Web of Science][Medline]
4. Durante M, Snigiryova G, Akaeva E, Bogomazova A, Druzhinin S, Fedorenko B, Greco O, Novitskaya N, Rubanovich A, Obe G. Chromosome aberration dosimetry in cosmonauts after single or multiple space flights. Cytogenet. Genome Res. (2003) 103:40–46.[CrossRef][Web of Science][Medline]
5. Scampoli P. Biological effects of accelerated protons. Radiother. Oncol. (2004) 73:S130–S133.[CrossRef][Web of Science][Medline]
6. Lin ZW, Adams JH Jr. Effects of nuclear cross sections at different energies on the radiation hazard from galactic cosmic rays. Radiat. Res. (2007) 167:330–337.[CrossRef][Web of Science][Medline]
7. Hellweg CE, Baumstark-Khan C. Getting ready for the manned mission to Mars: the astronauts’ risk from space radiation. Naturwissenschaften (2007) 94:517–526.[CrossRef][Web of Science][Medline]
8. Zhao Y, Johnsen R, Baillie D, Rose A. Worms in space? A model biological dosimeter. Gravit. Space Biol. Bull. (2005) 18:11–16.[Medline]
9. Hajek M, Berger T, Fugger M, Furstner M, Vana N, Akatov Y, Shurshakov V, Arkhangelsky V. Dose distribution in the Russian Segment of the International Space Station. Radiat. Prot. Dosimetry (2006) 120:446–449.
10. Ohnishi K, Ohnishi T. The biological effects of space irradiation during long stays in space. Biol. Sci. Space (2004) 18:201–205.[CrossRef][Medline]
11. Cucinotta FA, Kim M-HY, Ren L. Managing Lunar and Mars Mission Radiation Risks. Part 1: Cancer Risks, Uncertainties, and Sheilding Effectiveness (2005) Washington, DC: Nasa.
12. Nelson GA. Fundamental space radiobiology. Gravit. Space Biol. Bull. (2003) 16:29–36.[Medline]
13. Borak TB, Doke T, Fuse T, et al. Comparisons of LET distributions for protons with energies between 50 and 200 MeV determined using a spherical tissue-equivalent proportional counter (TEPC) and a position-sensitive silicon spectrometer (RRMD-III). Radiat. Res. (2004) 162:687–692.[CrossRef][Web of Science][Medline]
14. Kao J, Rosenstein BS, Peters S, Milano MT, Kron SJ. Cellular response to DNA damage. Ann. N. Y. Acad. Sci. (2005) 1066:243–258.[CrossRef][Web of Science][Medline]
15. Lieberman HB. Rad9, an evolutionarily conserved gene with multiple functions for preserving genomic integrity. J. Cell Biochem. (2006) 97:690–697.[CrossRef][Web of Science][Medline]
16. Gialanella G, Grossi GF, Macchiato MF, Napolitano M, Speranza PR. Contributions of various types of damage to inactivation of T4 bacteriophage by protons. Radiat. Res. (1983) 96:462–475.[CrossRef][Web of Science][Medline]
17. Frankenberg D, Brede HJ, Schrewe UJ, Steinmetz C, Frankenberg-Schwager M, Kasten G, Pralle E. Induction of DNA double-strand breaks in mammalian cells and yeast. Adv. Space Res. (2000) 25:2085–2094.[CrossRef][Web of Science][Medline]
18. Hada M, Sutherland BM. Spectrum of complex DNA damages depends on the incident radiation. Radiat. Res. (2006) 165:223–230.[CrossRef][Web of Science][Medline]
19. Kundrat P, Stewart RD. On the biophysical interpretation of lethal DNA lesions induced by ionising radiation. Radiat. Prot. Dosimetry (2006) 122:169–172.
20. Campa A, Ballarini F, Belli M, et al. DNA DSB induced in human cells by charged particles and gamma rays: experimental results and theoretical approaches. Int. J. Radiat. Biol. (2005) 81:841–854.[CrossRef][Web of Science][Medline]
21. Friedland W, Jacob P, Bernhardt P, Paretzke HG, Dingfelder M. Simulation of DNA damage after proton irradiation. Radiat. Res. (2003) 159:401–410.[CrossRef][Web of Science][Medline]
22. Goodhead DT. Initial events in the cellular effects of ionizing radiations: clustered damage in DNA. Int. J. Radiat. Biol. (1994) 65:7–17.[Web of Science][Medline]
23. Ward JF. The complexity of DNA damage: relevance to biological consequences. Int. J. Radiat. Biol. (1994) 66:427–432.[Web of Science][Medline]
24. Jakob B, Scholz M, Taucher-Scholz G. Biological imaging of heavy charged-particle tracks. Radiat. Res. (2003) 159:676–684.[CrossRef][Web of Science][Medline]
25. Desai N, Durante M, Lin ZW, Cucinotta F, Wu H. High LET-induced H2AX phosphorylation around the Bragg curve. Adv. Space Res. (2005) 35:236–242.[CrossRef][Web of Science][Medline]
26. Tartier L, Spenlehauer C, Newman HC, Folkard M, Prise KM, Michael BD, Menissier-deMurcia J, deMurcia G. Local DNA damage by proton microbeam irradiation induces poly(ADP-ribose) synthesis in mammalian cells. Mutagenesis (2003) 18:411–416.
27. Di Pietro C, Piro S, Tabbi G, et al. Cellular and molecular effects of protons: apoptosis induction and potential implications for cancer therapy. Apoptosis (2006) 11:57–66.[CrossRef][Web of Science][Medline]
28. Fry RC, Begley TJ, Samson LD. Genome-wide responses to DNA-damaging agents. Annu. Rev. Microbiol. (2005) 59:357–377.[CrossRef][Web of Science][Medline]
29. Beranek DT. Distribution of methyl and ethyl adducts following alkylation with monofunctional alkylating agents. Mutat. Res. (1990) 231:11–30.[Web of Science][Medline]
30. Xiao W, Chow BL, Rathgeber L. The repair of DNA methylation damage in Saccharomyces cerevisiae. Curr. Genet. (1996) 30:461–468.[CrossRef][Web of Science][Medline]
31. Hanna M, Chow BL, Morey NJ, Jinks-Robertson S, Doetsch PW, Xiao W. Involvement of two endonuclease III homologs in the base excision repair pathway for the processing of DNA alkylation damage in Saccharomyces cerevisiae. DNA Repair (2004) 3:51–59.[Medline]
32. Xiao W, Chow BL, Hanna M, Doetsch PW. Deletion of the MAG1 DNA glycosylase gene suppresses alkylation-induced killing and mutagenesis in yeast cells lacking AP endonucleases. Mutat. Res. (2001) 487:137–147.[Web of Science][Medline]
33. Tanaka T, Huang X, Halicka HD, Zhao H, Traganos F, Albino AP, Dai W, Darzynkiewicz Z. Cytometry of ATM activation and histone H2AX phosphorylation to estimate extent of DNA damage induced by exogenous agents. Cytometry A. (2007) 71:648–661.[Medline]
34. Broomfield S, Hryciw T, Xiao W. DNA postreplication repair and mutagenesis in Saccharomyces cerevisiae. Mutat. Res. (2001) 486:167–184.[Web of Science][Medline]
35. Krogh BO, Symington LS. Recombination proteins in yeast. Annu. Rev. Genet. (2004) 38:233–271.[CrossRef][Web of Science][Medline]
36. Dudasova Z, Dudas A, Chovanec M. Non-homologous end-joining factors of Saccharomyces cerevisiae. FEMS Microbiol. Rev. (2004) 28:581–601.[CrossRef][Web of Science][Medline]
37. Lewis LK, Resnick MA. Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae. Mutat. Res. (2000) 451:71–89.[Web of Science][Medline]
38. Ianzini F, Cherubini R, Mackey MA. Mitotic catastrophe induced by exposure of V79 Chinese hamster cells to low-energy protons. Int. J. Radiat. Biol. (1999) 75:717–723.[CrossRef][Web of Science][Medline]
39. Antoccia A, Sgura A, Cavinato M, Cherubini R, Tanzarella C. Cell cycle perturbations and cytogenetic damage induced by low energy protons in human primary fibroblasts. Radiat. Prot. Dosimetry (2002) 99:197–198.[Abstract]
40. Belli M, Cherubini R, Dalla Vecchia M, Dini V, Esposito G, Moschini G, Sapora O, Signoretti C, Simone G, Tabocchini MA. DNA fragmentation in mammalian cells exposed to various light ions. Adv. Space Res. (2001) 27:393–399.[CrossRef][Web of Science][Medline]
41. Belli M, Cherubini R, Dalla Vecchia M, Dini V, Esposito G, Moschini G, Sapora O, Simone G, Tabocchini MA. DNA fragmentation in V79 cells irradiated with light ions as measured by pulsed-field gel electrophoresis. I. Experimental results. Int. J. Radiat. Biol. (2002) 78:475–482.[CrossRef][Web of Science][Medline]
42. Adzuma K, Ogawa T, Ogawa H. Primary structure of the RAD52 gene in Saccharomyces cerevisiae. Mol. Cell. Biol. (1984) 4:2735–2744.
43. Leloup C, Garty G, Assaf G, et al. Evaluation of lesion clustering in irradiated plasmid DNA. Int. J. Radiat. Biol. (2005) 81:41–54.[Web of Science][Medline]
44. Harkness TAA, Davies GF, Ramaswamy V, Arnason TG. The ubiquitin-dependent targeting pathway in Saccharomyces cerevisiae plays a critical role in multiple chromatin assembly regulatory steps. Genetics (2002) 162:615–632.
45. Latif C, Elzen NR, O'Connell MJ. DNA damage checkpoint maintenance through sustained Chk1 activity. J. Cell Sci. (2004) 117:3489–3498.
46. Snow R. Mutants of yeast sensitive to ultraviolet light. J. Bacteriol. (1967) 94:571–575.
47. Nakai S, Matsumoto S. Two types of radiation-sensitive mutant in yeast. Mutat. Res. (1967) 4:129–136.[Web of Science][Medline]
48. Cox BS, Parry JM. The isolation, genetics and survival characteristics of ultraviolet light-sensitive mutants in yeast. Mutat. Res. (1968) 6:37–55.[Web of Science][Medline]
49. Resnick MA. Induction of mutations in Saccharomyces cerevisiae by ultraviolet light. Mutat. Res. (1969) 7:315–332.[Web of Science][Medline]
50. Game JC, Mortimer RK. A genetic study of x-ray sensitive mutants in yeast. Mutat. Res. (1974) 24:281–292.[CrossRef][Web of Science][Medline]
51. Moore CW. Responses of radiation-sensitive mutants of Saccharomyces cerevisiae to lethal effects of bleomycin. Mutat. Res. (1978) 51:165–180.[Web of Science][Medline]
52. Geigl EM, Eckardt-Schupp F. Repair of gamma ray-induced S1 nuclease hypersensitive sites in yeast depends on homologous mitotic recombination and a RAD18-dependent function. Curr. Genet. (1991) 20:33–37.[CrossRef][Web of Science][Medline]
53. Krogh BO, Symington LS. Recombination proteins in yeast. Annu. Rev. Genet. (2004) 38:233–271.[CrossRef][Web of Science][Medline]
54. Kiefer J, Egenolf R, Ikpeme S. Heavy ion-induced DNA double-strand breaks in yeast. Radiat. Res. (2002) 157:141–148.[CrossRef][Web of Science][Medline]
55. Yamashita YM, Okada T, Matsusaka T, Sonoda E, Zhao GY, Araki K, Tateishi S, Yamaizumi M, Takeda S. RAD18 and RAD54 cooperatively contribute to maintenance of genomic stability in vertebrate cells. EMBO J. (2002) 21:5558–5566.[CrossRef][Web of Science][Medline]
56. Sonoda E, Hochegger H, Saberi A, Taniguchi Y, Takeda S. Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair (2006) 5:1021–1029.[Medline]
57. Symington LS. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol. Mol. Biol. Rev. (2002) 66:630–670.
58. Zou H, Rothstein R. Holliday junctions accumulate in replication mutants via a RecA homolog-independent mechanism. Cell (1997) 90:87–96.[CrossRef][Web of Science][Medline]
59. McKee RH, Lawrence CW. Genetic analysis of gamma-ray mutagenesis in yeast. I. Reversion in radiation-sensitive strains. Genetics (1979) 93:361–373.
60. von Borstel RC, Cain KT, Steinberg CM. Inheritance of spontaneous mutability in yeast. Genetics (1971) 69:17–27.
61. Quah SK, von Borstel RC, Hastings PJ. The origin of spontaneous mutation in Saccharomyces cerevisiae. Genetics (1980) 96:819–839.
62. Roche H, Gietz RD, Kunz BA. Specificities of the Saccharomyces cerevisiae rad6, rad18, and rad52 mutators exhibit different degrees of dependence on the REV3 gene product, a putative nonessential DNA polymerase. Genetics (1995) 140:443–456.[Abstract]
63. Huang ME, de Calignon A, Nicolas A, Galibert F. POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase delta, defines a link between DNA replication and the mutagenic bypass repair pathway. Curr. Genet. (2000) 38:178–187.[CrossRef][Web of Science][Medline]
64. Aboussekhra A, Chanet R, Zgaga Z, Cassier-Chauvat C, Heude M, Fabre F. RADH, a gene of Saccharomyces cerevisiae encoding a putative DNA helicase involved in DNA repair. Characteristics of radH mutants and sequence of the gene. Nucleic Acids Res. (1989) 17:7211–7219.
65. Stelter P, Ulrich HD. Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature (2003) 425:188–191.[CrossRef][Medline]
Received on May 30, 2007; revised on December 15, 2007; accepted on December 18, 2007.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||