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Department of Applied Microbiology and Food Science, College of Agriculture, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5A8 Canada
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ABSTRACT |
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Ultraviolet (UV) radiation-induced DNA damage leading to entomopathogenic fungal inactivation is commonly measured by viability counts. Here we report the first quantification of UV-induced cyclobutane pyrimidine dimers (CPD) in DNA of the entomopathogenic fungus, Beauveria bassiana. Changes in the mobility of UV-C irradiated DNA were resolved with CPD specific bacteriophage T4 endonuclease V and alkaline agarose gel electrophoresis. The maximum number of CPD formed in B. bassiana DNA in vitro by UV-C irradiation was 28 CPD/ 10 kb after 720 J/m2 dose. The maximum number of CPDs formed in B. bassiana conidiospore DNA irradiated in vivo was 15 CPD/10 kb after 480 J/m2 dose and was quantified from conidiospores that were incubated to allow photoreactivation and nucleotide excision repair. The conidiospores incubated for photoreactivation and nucleotide excision repair showed decreased number of CPD/10 kb DNA and a higher percent survival of conidiospore populations than conidiospores not allowed to repair.
Key words: DNA damage, DNA repair, entomopathogenic fungi, T4 endonuclease V, ultraviolet irradiation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Early work found that B. bassiana conidiospores exposed to sunlight caused less mortality in insects (Gardner et al 1977), and experiments with simulated and natural sunlight also showed similar outcomes including decreased conidiospore viability and germination (Inglis et al 1997; Inglis et al 1993, 1995; Morley-Davies et al 1995). Radiation at UV-B (280–320 nm) wavelengths from natural or simulated sunlight is the primary photochemical reaction that damages DNA and affects survival or percent germination (Inglis et al 1995, Costa et al 2001, Ravanat et al 2001). Under laboratory conditions UV-C (254 nm) sometimes is used because the generated photochemical reactions are more efficient since UV-C is the closest to the absorption spectrum of the pyrimidine bases (Ravanat et al 2001). Its physiological effects on EPF are largely the same as UV-B (Varela and Morales 1996, Hu et al 1996).
In a DNA molecule where pyrimidine bases are adjacent to each other two main types of dimers are formed: (i) cyclobutane pyrimidine dimer (CPD), which accounts for 70% of the DNA damage incurred from UV-B/UV-C irradiation; and (ii) pyrimidine-(6-4)-pyrimidone photoproduct ([6-4]PP) (Wang 1976). The use of UV-C radiation induces the formation of (6-4)PP at a level that represents 20–30% of the total UV dimers (Wang 1976), but the (6-4)PP produced by simulated natural sunlight could be 10% (Yoon et al 2000).
Inactivation of the conidiospores occurs when DNA repair pathways cannot mend the UV-induced damage and the conidiospore dies. The two pathways that mend CPD are photoreactivation and nucleotide-excision repair (NER) (Friedberg et al 1995). An excision repair system is required in most organisms to mend (6-4)PP (Friedberg et al 1995). There are also (6-4)PP photolyases (Todo et al 1993, Yasui and Eker 1998), but these have not been identified in filamentous fungi (Chelico et al 2003, Borkovich et al 2004, Goldman and Kafer 2004). Here we define photoreactivation as the classical CPD photolyase (Kelner 1949). Photoreactivation, a two-step repair mechanism carried out by a photolyase, monomerizes UV-induced pyrimidine-pyrimidine dimers upon illumination of the photolyase with visible light at 380–410 nm (Yasui and Eker 1998). The NER pathway, on the other hand, is the most versatile mechanism of DNA damage repair where CPD and (6-4)PP are removed as part of a 30-nucleotide long bimodal excision. The subsequent gap in the DNA strand is filled in by a DNA polymerase that uses the complementary strand as a template (Friedberg et al 1995).
Although studies with B. bassiana and other EPF have purported or alluded to DNA damage and DNA repair being responsible for fungal inactivation there has been no direct evidence of DNA damage (Hunt et al 1994, Alves et al 1998, Braga et al 2001, Gilberto et al 2001, Braga et al 2002). The objective of the present study was to determine the formation of the primary UV photoproduct, CPD, in B. bassiana DNA and repair of these lesions by photoreactivation and NER. This is the first study to quantify DNA damage in an EPF.
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MATERIALS AND METHODS |
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-pAJ. The plasmid, pAJ (2.9 kb pTZ18R, GenBank accession number L08956
[GenBank]
), containing an insert constructed of 24 thymine residues was created by ligating the construct into the BamHI/EcoRI digested plasmid. The E. coli DH5-pAJ cultures were grown at 37 C for 16–24 h. The broth cultures were rotated in a water bath at 150 rpm.
UV irradiation procedures.— –
A 30 or 5 mL conidiospore suspension in 5% (w/v) glucose and 0.02% (v/v) Tween-80 was adjusted to a concentration of 1 x 108 conidiospores/ mL. Concentrations were determined with a haemocytometer. The addition of Tween-80 prevents conidiospores from aggregating during UV irradiation thereby decreasing the chance that conidiospores may shield a segment of the population from UV light. The freshly prepared suspensions were placed in glass Petri dishes (surface area = 64 cm2 or 4.5 cm2) and exposed to ultraviolet radiation (UVP Miner-alight R52G; Upland, California) between 200 and 280 nm (
max = 254 nm) at an irradiance of 0.4 W/m2 as determined by a Blak-Ray® Ultraviolet Intensity Meter model J225 (San Gabriel, California). The suspension was stirred continuously with a magnetic stirrer and stir bar. These conditions allowed random irradiation of the conidiospores. Irradiation was conducted to promote NER and enzymatic photoreactivation in B. bassiana and consisted of irradiation in the presence of fluorescent light (0.7 W/m2). The temperature during irradiation was 25 C. Samples of 1 mL were taken at appropriate intervals during irradiation. Samples were treated post-irradiation in two ways. First, samples representing no DNA repair were processed immediately after irradiation by dilution and plating onto Y PGA for enumeration of survival or isolation of the DNA for CPD quantification. Second, samples used to study DNA repair were incubated immediately for repair with these conditions: The temperature of repair incubation was 27 C; the 6 h repair incubation was conducted to promote NER and enzymatic photoreactivation in B. bassiana by incubation of samples 60 cm from two fluorescent (34 W, Sylvania F40T12/CW/SS) and two halogen (60 W, Sylvania) light sources resulting in an irradiance of 1.29 W/m2 as determined by a Line Quantum Sensor, Model LI-250 Light Meter (Lincoln, Nebraska).
Determination of conidiospore viability.— – Tenfold dilution of samples was made and drop plates prepared by applying four 25 µL drops to Y PGA, in triplicate. Drop plates were incubated in the dark at 27 C and colonies were counted after 3, 4 and 5 d. Plate counts were equated to colony-forming units (CFU) per mL.
Isolation and extraction of DNA.— –
Beauveria bassiana conidiospore DNA was isolated with the small-scale glass bead method as described in Chelico and Khachatourians (2003). The plasmid pAJ was isolated from E. coli DH5 as described in Sambrook and Russel (2001).
UV irradiation of DNA in vitro.— –
DNA samples from B. bassiana and a plasmid pAJ, isolated from E. coli DH5 were irradiated in vitro. The DNA irradiated was standardized at 3 µg. The DNA concentration was determined with a spectrophotometer (Sambrook and Russel 2001) and visually. For visual determination of DNA concentration a 1 µL aliquot of the DNA sample was resolved on a 1% (w/v) aga-rose gel with 1 µg of lambda phage DNA digested with HindIII or a DNA sample of known concentration. This relative determination of DNA concentration also served as a quality assurance measure to confirm the integrity of DNA sample before UV irradiation. The volumes irradiated were equalized to 10 µL with deionized distilled water and dropped onto parafilm. The pAJ samples were treated with ScaI followed by phenol : chloroform extraction to remove contaminating protein before irradiation. This preliminary step linearized the plasmid. The CPD formed in the plasmid DNA therefore were quantified as CPD per 2.9 kb. The DNA samples in these 10 µL aliquots were exposed to ultraviolet light (UVP Mineralight R52G) between 200 and 280 nm (max = 254 nm) at an irradiance of 0.4 W/m2 as determined by a Blak-Ray® Ultraviolet Intensity Meter model J225.
Quantification of CPD formation.— –
DNA was digested with 1.8–3.6 units T4 endonuclease V (Worthington Biochemical Corp., Lakewood, New Jersey [product discontinued], or Epicentre Technologies, Madison, Wisconsin) and 8 µL of reaction buffer (0.01 mM ethylenediaminetetraacetic acid, 0.04 mM NaPO4, pH 6.5) at least 1 h and up to 24 h at 37 C. In each experiment duplicate DNA samples were separated on 1% (w/v) alkaline agarose gels by alkaline gel electrophoresis. The method described by Drouin et al (1996) was followed. The digital images of alkaline gels were analyzed with an Alpha Innotech Corp. IS-1000 Digital Imaging System (San Leandro, California). Densitometry analysis was performed for each lane. Integration was used to determine the area of the peak DNA migration, which was used to calculate the break frequency. Calculation of CPD formed per 10 kb of DNA was performed according to Sutherland et al (1996), a method based on break frequency of DNA. This analysis requires that a control DNA sample be used to determine the total size of the T4 endonuclease V digested DNA sample. Control DNA samples were irradiated and resolved by alkaline gel electrophoresis without T4 endonuclease V treatment. The small-scale DNA extraction method gives a reproducible size of B. bassiana genomic DNA. The theory behind this analysis is presented in detail by Sutherland et al (1996). Briefly, the average length of the DNA population will decrease after T4 endonuclease V treatment of UV-irradiated DNA. The breaks per unit length of DNA, here we use per 10 kb, is calculated by subtracting the inverse of the final break frequency from the inverse of the initial break frequency (Sutherland et al 1996). An additional negative control was to treat unirradiated DNA with T4 endonuclease V, which ensured that no nonspecific nicks were introduced to the DNA.
Statistical analysis.— – Percent survival and CPD values were obtained from three or more independent experiments. The results of each experiment were examined with one way completely randomized analysis of variance to determine which experiments were not significantly different. The CoStat, version 6.204 program (CoHort Software, Monterey, California) was used. Results that were not significantly different were combined and averaged. The standard deviation of the mean of the three (or more) experiments was calculated and used for comparison to different treatments.
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RESULTS |
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) and analysis is presented (TABLE I). The controls in Lane 1 (FIG. 1A, B) represent the initial break frequency used in the calculation of the CPD/10 kb (Sutherland et al 1996). T4 endonuclease V specificity was checked by testing the ability of the enzyme to introduce nonspecific nicks in unir-radiated DNA. We found the T4 endonuclease V to be specific, and it did not introduce any nonspecific nicks in the DNA (results not shown). The maximum value of CPD/10 kb for pAJ was 12 ± 2 CPD/2.9 kb. Extended enzyme treatment beyond 1 h did not alter the value of CPD in pAJ or B. bassiana samples. The method employed is specific and sensitive enough to quantify CPD values in the DNA over a significantly different, low and high UV dose (pAJ) and was not affected by DNA concentration (B. bassiana) (TABLE I). At 720 J/m2 B. bassiana DNA irradiated in vitro has an average of 28 CPD/10 kb.
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). The formation of CPD was linear but saturated at ca. 720 J/ m2 UV dose (FIG. 2B). The 720 J/m2 dose (FIG. 2A, Lane 8) is representative of the 960, 1200 and 1440 J/m2 doses. The B. bassiana CPD maximum of 28 CPD/10 kb has been confirmed in our lab for six strains other than the strain in this study, GK2016 (results not shown).
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). The control for no repair was extraction of DNA from conidiospores immediately after irradiation and treatment with T4 endonuclease V (FIG. 3B). Comparison of conditions promoting photoreactivation and that which minimize photoreactivation gives an estimate of the removal of CPD from B. bassiana due to repair mechanisms (FIG. 3C). The CPD quantified is less in DNA isolated from conidiospores allowed to conduct DNA repair. The conidiospores not incubated for repair show CPD initially formed in the DNA. When photoreactivation and NER are promoted the CPD formation in conidiospores is linear for approximately the first 360 J/m2 of irradiation and saturates at ca. 14–15 CPD/10 kb of DNA. In support of the data that the decreased number of CPD/10 kb quantified from DNA isolated from B. bassiana conidiospores incubated 6 h is due to repair, the survival of the conidiospores under these conditions also was determined (FIG. 3D). This type of survival experiment is a classical method of studying DNA repair (Friedberg 1988). The conidiospores incubated for repair show a higher percent survival than conidiospore populations not incubated for repair.
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DISCUSSION |
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; Alves et al 1998; Braga et al 2001; Gilberto et al 2001; Braga et al 2002; Gardner et al 1977; Inglis et al 1993, 1995; Inglis et al 1997; Morely-Davies et al 1995; Costa et al 2001). Here we show direct proof that it is UV-C irradiation of B. bassiana DNA that causes the formation of CPD and loss of viability (FIGS. 2
and 3).
Our experiments confirmed that T4 endonuclease V analysis of UV-damaged DNA (Yasuda and Sekiguchi 1970) is specific and accurate. The results of estimation of CPD in pAJ DNA ((TABLE I) show that varying UV dose causes a discernable difference in the number of CPD and their quantification is unaffected by the duration of enzymatic treatment. Similarly, varying DNA concentration of B. bassiana showed that it had no limiting effect on CPD quantification.
Formation of CPD also was quantified from in vitro-irradiated B. bassiana DNA. It is known that CPD formation is more efficient between two adjacent thymines than thymine-cytosine, cytosine-thymine or cytosine-cytosine (Ravanat et al 2001). The theoretical maximum number of CPD may never form in DNA due to the sequence preference of CPD formation at thymine-thymine. Therefore, although the maximum CPD/10 kb we found for B. bassiana saturated at 28 CPD/10 kb, it does not mean that all potential CPD sites in the DNA sequence were dimerized.
The maximum CPD formation in conidiospore DNA of B. bassiana, is 15 per 10 kb and levels off after ca. 480 J/m2 UV dose, which coincides with the time when the maximum numbers of conidiospores have been killed by irradiation (FIG. 3C, D). Although (6-4)PP also form in DNA after UV-C irradiation these lesions cannot be quantified by alkaline gel electrophoresis. However, (6-4)PP lesions can be mended also by NER during repair incubation (Chelico et al 2003). The correlation (FIG. 3C, D) between survival and CPD formation suggests that UV induced photoproducts in the DNA are a major cause of B. bassiana conidiospore killing. This is consistent with other studies indicating CPD formation represents 70% of the DNA damage induced by irradiation (Wang 1976).
The maximum number of CPD formed in B. bassiana DNA irradiated in vivo if repair was prevented is 28 CPD/10 kb, and this occurred after 480 J/m2 of UV exposure (FIG. 3C). However the maximum number of CPD formed in B. bassiana DNA irradiated in vivo if allowed to repair CPD by photoreactivation was 15 CPD/10 kb and occurred after 480 J/ m2 of UV exposure (FIG. 3C). This difference indicates a significant number of dimers formed in DNA irradiated in vivo are removed through their conversion to monomers by the repair system. The accumulated CPD in B. bassiana conidiospores levels off of after 480 J/m2 UV dose (FIG. 3C). This might be because the few surviving conidiospores still are able to repair some DNA lesions and explains why the number CPD/10 kb does not continue to increase when the conidiospore viability has reached 0.003% (FIG. 3D).
Our study reports the first demonstration of number of CPD quantified per 10 kb of UV-exposed EPF DNA. Although UV radiation was the first environmental constraint on EPF to be identified (Ignoffo et al 1977, Fargues 2003), no other quantitative studies of DNA damage in EPF have been conducted. We have shown quantitatively that UV-C irradiation of B. bassiana causes formation of CPD and loss of conidiospore viability. There can be three broader uses of our findings on (i) timing of release of a fungal insecticide (e.g. sunny or cloudy days), (ii) formulations that would mitigate UV induced DNA damage (Cantrell et al 2001) and (iii) screening UV-tolerant isolates.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. E-mail: george.khachatourians{at}usask.ca
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LITERATURE CITED |
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X174HaeIII markers with sizes (kb) of relevant bands shown in the margin.
