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Mycologia, 97(3), 2005, pp. 621627.q 2005 by The Mycological Society of America, Lawrence, KS 66044-8897

Quantification of ultraviolet-C irradiation induced cyclobutane pyrimidinedimers and their removal in Beauveria bassiana conidiospore DNA

Linda ChelicoJanna L. HaughianAdrienne E. WoytowichGeorge G. Khachatourians1

Department of Applied Microbiology and Food Science,College of Agriculture, University of Saskatchewan,Saskatoon, Saskatchewan, S7N 5A8 Canada

Abstract: Ultraviolet (UV) radiation-induced DNAdamage leading to entomopathogenic fungal inacti-vation is commonly measured by viability counts.Here we report the first quantification of UV-inducedcyclobutane pyrimidine dimers (CPD) in DNA of theentomopathogenic fungus, Beauveria bassiana.Changes in the mobility of UV-C irradiated DNA wereresolved with CPD specific bacteriophage T4 endo-nuclease V and alkaline agarose gel electrophoresis.The maximum number of CPD formed in B. bassi-ana DNA in vitro by UV-C irradiation was 28 CPD/10 kb after 720 J/m2 dose. The maximum numberof CPDs formed in B. bassiana conidiospore DNAirradiated in vivo was 15 CPD/10 kb after 480 J/m2

dose and was quantified from conidiospores thatwere incubated to allow photoreactivation and nucle-otide excision repair. The conidiospores incubatedfor photoreactivation and nucleotide excision repairshowed decreased number of CPD/10 kb DNA anda higher percent survival of conidiospore popula-tions than conidiospores not allowed to repair.

Key words: DNA damage, DNA repair, entomo-pathogenic fungi, T4 endonuclease V, ultraviolet ir-radiation

INTRODUCTION

The effect of ultraviolet (UV) radiation on the en-tomopathogenic fungus (EPF) Beauveria bassianahas been studied in natural and laboratory environ-ments (Feng et al 1994). Early work found that B.bassiana conidiospores exposed to sunlight causedless mortality in insects (Gardner et al 1977), andexperiments with simulated and natural sunlight alsoshowed similar outcomes including decreased conid-iospore viability and germination (Inglis et al 1997;

Accepted for publication 15 Mar 2005.1 Corresponding author. E-mail: george.khachatourians@usask.ca

Inglis et al 1993, 1995; Morley-Davies et al 1995). Ra-diation at UV-B (280320 nm) wavelengths from nat-ural or simulated sunlight is the primary photochem-ical reaction that damages DNA and affects survivalor percent germination (Inglis et al 1995, Costa et al2001, Ravanat et al 2001). Under laboratory condi-tions UV-C (254 nm) sometimes is used because thegenerated photochemical reactions are more effi-cient since UV-C is the closest to the absorption spec-trum of the pyrimidine bases (Ravanat et al 2001).Its physiological effects on EPF are largely the sameas UV-B (Varela and Morales 1996, Hu et al 1996).

In a DNA molecule where pyrimidine bases are ad-jacent to each other two main types of dimers areformed: (i) cyclobutane pyrimidine dimer (CPD),which accounts for 70% of the DNA damage in-curred from UV-B/UV-C irradiation; and (ii) pyrim-idine-(6-4)-pyrimidone photoproduct ([6-4]PP)(Wang 1976). The use of UV-C radiation induces theformation of (6-4)PP at a level that represents 2030% of the total UV dimers (Wang 1976), but the (6-4)PP produced by simulated natural sunlight couldbe 10% (Yoon et al 2000).

Inactivation of the conidiospores occurs whenDNA repair pathways cannot mend the UV-induceddamage and the conidiospore dies. The two pathwaysthat mend CPD are photoreactivation and nucleo-tide-excision repair (NER) (Friedberg et al 1995). Anexcision repair system is required in most organismsto mend (6-4)PP (Friedberg et al 1995). There arealso (6-4)PP photolyases (Todo et al 1993, Yasui andEker 1998), but these have not been identified infilamentous fungi (Chelico et al 2003, Borkovich etal 2004, Goldman and Kafer 2004). Here we definephotoreactivation as the classical CPD photolyase(Kelner 1949). Photoreactivation, a two-step repairmechanism carried out by a photolyase, monomer-izes UV-induced pyrimidine-pyrimidine dimers uponillumination of the photolyase with visible light at380410 nm (Yasui and Eker 1998). The NER path-way, on the other hand, is the most versatile mecha-nism of DNA damage repair where CPD and (6-4)PPare removed as part of a 30-nucleotide long bimodalexcision. The subsequent gap in the DNA strand isfilled in by a DNA polymerase that uses the comple-mentary strand as a template (Friedberg et al 1995).

Although studies with B. bassiana and other EPF

622 MYCOLOGIA

have purported or alluded to DNA damage and DNArepair being responsible for fungal inactivation therehas been no direct evidence of DNA damage (Huntet al 1994, Alves et al 1998, Braga et al 2001, Gilbertoet al 2001, Braga et al 2002). The objective of thepresent study was to determine the formation of theprimary UV photoproduct, CPD, in B. bassiana DNAand repair of these lesions by photoreactivation andNER. This is the first study to quantify DNA damagein an EPF.

MATERIALS AND METHODS

Microbial isolates and culture conditions.Beauveria bassi-ana GK2016 (BioInsecticide Research Laboratory, Depart-ment of Applied Microbiology and Food Science, Collegeof Agriculture, University of Saskatchewan, Canada) was cul-tured on Y PG agar (Y PGA) at 27 C for 7 d. Conidiosporesfrom a culture were harvested by flooding the plate withdeionized distilled water, dislodging of conidiospores witha bent glass rod and passage through a glass wool filter toremove mycelia. Conidiospores were recovered by centri-fugation at 4000 3 g for further use. Luria-Bertani agar (10g/L bactotryptone, 5 g/L yeast extract, 10 g/L NaCl, 20g/L agar, pH 7.5) or broth containing 50 mg/mL ampicillinwas inoculated from frozen stocks of Escherichia coli DH5a-pAJ. The plasmid, pAJ (2.9 kb pTZ18R, GenBank accessionnumber L08956), containing an insert constructed of 24thymine residues was created by ligating the construct intothe BamHI/EcoRI digested plasmid. The E. coli DH5a-pAJcultures were grown at 37 C for 1624 h. The broth cultureswere rotated in a water bath at 150 rpm.

UV irradiation procedures.A 30 or 5 mL conidiospore sus-pension in 5% (w/v) glucose and 0.02% (v/v) Tween-80was adjusted to a concentration of 1 3 108 conidiospores/mL. Concentrations were determined with a haemocytom-eter. The addition of Tween-80 prevents conidiospores fromaggregating during UV irradiation thereby decreasing thechance that conidiospores may shield a segment of the pop-ulation from UV light. The freshly prepared suspensionswere placed in glass Petri dishes (surface area 5 64 cm2 or4.5 cm2) and exposed to ultraviolet radiation (UVP Miner-alight R52G; Upland, California) between 200 and 280 nm(lmax 5 254 nm) at an irradiance of 0.4 W/m2 as deter-mined by a Blak-Rayt Ultraviolet Intensity Meter modelJ225 (San Gabriel, California). The suspension was stirredcontinuously with a magnetic stirrer and stir bar. These con-ditions allowed random irradiation of the conidiospores. Ir-radiation was conducted to promote NER and enzymaticphotoreactivation in B. bassiana and consisted of irradia-tion in the presence of fluorescent light (0.7 W/m2). Thetemperature during irradiation was 25 C. Samples of 1 mLwere taken at appropriate intervals during irradiation. Sam-ples were treated post-irradiation in two ways. First, samplesrepresenting no DNA repair were processed immediatelyafter irradiation by dilution and plating onto Y PGA for enu-meration of survival or isolation of the DNA for CPD quan-tification. 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 hrepair incubation was conducted to promote NER and en-zymatic photoreactivation in B. bassiana by incubation ofsamples 60 cm from two fluorescent (34 W, SylvaniaF40T12/CW/SS) and two halogen (60 W, Sylvania) lightsources resulting in an irradiance of 1.29 W/m2 as deter-mined by a Line Quantum Sensor, Model LI-250 Light Me-ter (Lincoln, Nebraska).

Determination of conidiospore viability.Tenfold dilution ofsamples was made and drop plates prepared by applyingfour 25 mL drops to Y PGA, in triplicate. Drop plates wereincubated in the dark at 27 C and colonies were countedafter 3, 4 and 5 d. Plate counts were equated to colony-forming units (CFU) per mL.

Isolation and extraction of DNA.Beauveria bassiana conid-iospore DNA was isolated with the small-scale glass beadmethod as described in Chelico and Khachatourians(2003). The plasmid pAJ was isolated from E. coli DH5a asdescribed in Sambrook and Russel (2001).

UV irradiation of DNA in vitro.DNA samples from B. bas-siana and a plasmid pAJ, isolated from E. coli DH5a wereirradiated in vitro. The DNA irradiated was standardized at3 mg. The DNA concentration was determined with a spec-trophotometer (Sambrook and Russel 2001) and visually.For visual determination of DNA concentration a 1 mL ali-quot of the DNA sample was resolved on a 1% (w/v) aga-rose gel with 1 mg of lambda phage DNA digested withHindIII or a DNA sample of known concentration. This rel-ative determination of DNA concentration also served as aquality assurance measure to confirm the integrity of DNAsample before UV irradiation. The volumes irradiated wereequalized to 10 mL with deionized distilled water anddropped onto parafilm. The pAJ samples were treated withScaI followed by phenol : chloroform extraction to removecontaminating protein before irradiation. This preliminarystep linearized the plasmid. The CPD formed in the plas-mid DNA therefore were quantified as CPD per 2.9 kb. TheDNA samples in these 10 mL aliquots were exposed to ul-traviolet light (UVP Mineralight R52G) between 200 and280 nm (lmax 5 254 nm) at an irradiance of 0.4 W/m2 asdetermined by a Blak-Rayt Ultraviolet Intensity Meter mod-el J225.

Quantification of CPD formation.DNA was digested with1.83.6 units T4 endonuclease V (Worthington BiochemicalCorp., Lakewood, New Jersey [product discontinued], orEpicentre Technologies, Madison, Wisconsin) and 8 mL ofreaction buffer (0.01 mM ethylenediaminetetraacetic acid,0.04 mM NaPO4, pH 6.5) at least 1 h and up to 24 h at 37C. In each experiment duplicate DNA samples were sepa-rated on 1% (w/v) alkaline agarose gels by alkaline gel elec-trophoresis. The method described by Drouin et al (1996)was followed. The digital images of alkaline gels were ana-lyzed with an Alpha Innotech Corp. IS-1000 Digital ImagingSystem (San Leandro, California). Densitometry analysiswas performed for each lane. Integration was used to de-termine the area of the peak DNA migration, which wasused to calculate the break frequency. Calculation of CPD

623CHELICO ET AL: ULTRAVIOLET DAMAGE IN BEAUVERIA BASSIANA

FIG. 1. Ethidium bromide-stained alkaline agarose gelsof UV irradiated pAJ and B. bassiana DNA digested withT4 endonuclease V. A. Lanes 13, pAJ DNA UV irradiated(representative of 12 and 720 J/m2) but not treated withT4 endonuclease V (control), pAJ UV irradiated 12 J/m2,and pAJ UV irradiated 720 J/m2, respectively. B. Lanes 13, B. bassiana DNA UV irradiated (720 J/m2) but not treat-ed with T4 endonuclease V (control), 13 and 23 B. bas-siana DNA UV irradiated 720 J/m2, respectively. Lanes Mare lHindIII/fX174HaeIII markers with sizes (kb) of rel-evant bands shown in the margin.

TABLE I. Formation of CPD in DNA UV irradiated in vitro

DNA sourceUV dose( J/m2)

Treatment(h)a CPD/unitb

pAJc 12 1.53.0

24

657

720 1.53.0

24

121111

B. bassianad

1X samplee 720 1.53.0

24

252627

2X samplee 720 1.524

2526

a T4 endonuclease V enzymatic treatment.b Unit values are per 2.9 kb for pAJ and per 10 kb for B.

bassiana.c The standard deviation of the mean CPD/unit, based on

three trials is 6 2.d The standard deviation of the mean CPD/unit, based

on three trials is 6 3.e The B. bassiana samples referred to as 1X and 2X cor-

respond to DNA concentrations of 3 mg (standard concen-tration used in experiments) and 6 mg, respectively.

formed per 10 kb of DNA was performed according to Suth-erland et al (1996), a method based on break frequency ofDNA. This analysis requires that a control DNA sample beused to determine the total size of the T4 endonuclease Vdigested DNA sample. Control DNA samples were irradiat-ed and resolved by alkaline gel electrophoresis without T4endonuclease V treatment. The small-scale DNA extractionmethod gives a reproducible size of B. bassiana genomicDNA. The theory behind this analysis is presented in detailby Sutherland et al (1996). Briefly, the average length ofthe DNA population will decrease after T4 endonuclease Vtreatment of UV-irradiated DNA. The breaks per unitlength of DNA, here we use per 10 kb, is calculated by sub-tracting the inverse of the final break frequency from theinverse of the initial break frequency (Sutherland et al1996). An additional negative control was to treat unirra-diated DNA with T4 endonuclease V, which ensured thatno nonspecific nicks were introduced to the DNA.

Statistical analysis.Percent survival and CPD values wereobtained from three or more independent experiments.The results of each experiment were examined with oneway completely randomized analysis of variance to deter-mine which experiments were not significantly different.The CoStat, version 6.204 program (CoHort Software, Mon-terey, California) was used. Results that were not signifi-cantly different were combined and averaged. The standarddeviation of the mean of the three (or more) experimentswas calculated and used for comparison to different treat-ments.

RESULTS

Quantification of CPD in DNA UV irradiated in vi-tro.The ability of T4 endonuclease V to make sin-

gle-stranded cuts at CPD sites was used to calculatethe occurrence of CPD in the irradiated pAJ and B.bassiana DNA. Representatives of resultant gels areshown (FIG. 1) and analysis is presented (TABLE I).The controls in Lane 1 (FIG. 1A, B) represent theinitial break frequency used in the calculation of theCPD/10 kb (Sutherland et al 1996). T4 endonucle-ase V specificity was checked by testing the ability ofthe enzyme to introduce nonspecific nicks in unir-radiated DNA. We found the T4 endonuclease V tobe specific, and it did not introduce any nonspecificnicks in the DNA (results not shown). The maximumvalue of CPD/10 kb for pAJ was 12 6 2 CPD/2.9 kb.Extended enzyme treatment beyond 1 h did not alterthe value of CPD in pAJ or B. bassiana samples. Themethod employed is specific and sensitive enough toquantify CPD values in the DNA over a significantlydifferent, low and high UV dose (pAJ) and was notaffected by DNA concentration (B. bassiana) (TABLEI). At 720 J/m2 B. bassiana DNA irradiated in vitrohas an average of 28 CPD/10 kb.

Purified B. bassiana DNA was exposed to 01080J/m2 radiation. An example of an ethidium bromide-stained gel of irradiated B. bassiana DNA digestedwith T4 endonuclease V is shown (FIG. 2A). The for-mation 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 1440J/m2 doses. The B. bassiana CPD maximum of 28

624 MYCOLOGIA

FIG. 2. Formation of CPD after in vitro exposure of B. bassiana DNA to UV-C radiation. A. Ethidium bromide-stainedalkaline agarose gel of B. bassiana DNA digested with T4 endonuclease V after exposure to varying doses of UV. Lanes: 1,DNA UV irradiated (120 J/m2) but not treated with T4 endonuclease V (control); 2, DNA not UV irradiated but treatedwith T4 endonuclease V (control, 0 J/m2); 38, 120, 240, 360, 480, 600 and 720 J/m2, respectively. Lane M is lHindIII/fX174HaeIII marker with sizes (kb) of relevant bands shown in the right margin. B. The number of CPD formed per 10 kbof DNA as a function of UV dose. The curve was constructed from the average of three independent experiments with barsreflecting the standard deviation of the mean.

CPD/10 kb has been confirmed in our lab for sixstrains other than the strain in this study, GK2016(results not shown).

CPD formation in DNA extracted from UV irradiated B.bassiana.CPD in B. bassiana conidiospores was de-termined after DNA repair conducted under condi-tions that promoted both photoreactivation andNER. This meant the DNA was isolated after the UV-irradiated conidiospores were incubated in the pres-ence of photoreactivating light for 6 h (FIG. 3A). Thecontrol for no repair was extraction of DNA fromconidiospores immediately after irradiation andtreatment with T4 endonuclease V (FIG. 3B). Com-parison of conditions promoting photoreactivationand that which minimize photoreactivation gives anestimate of the removal of CPD from B. bassiana dueto repair mechanisms (FIG. 3C). The CPD quantifiedis less in DNA isolated from conidiospores allowed toconduct DNA repair. The conidiospores not incubat-ed for repair show CPD initially formed in the DNA.When photoreactivation and NER are promoted theCPD formation in conidiospores is linear for approx-imately the first 360 J/m2 of irradiation and saturatesat ca. 1415 CPD/10 kb of DNA. In support of thedata that the decreased number of CPD/10 kb quan-tified from DNA isolated from B. bassiana conidio-spores incubated 6 h is due to repair, the survival of

the conidiospores under these conditions also was de-termined (FIG. 3D). This type of survival experimentis a classical method of studying DNA repair (Fried-berg 1988). The conidiospores incubated for repairshow a higher percent survival than conidiosporepopulations not incubated for repair.

DISCUSSION

The primary effects associated with solar radiation onEPF are decreased ability to cause insect mortality,delayed germination and ultimately inactivation orloss of viability. Studies on reduction in viability andgermination or decreases in infectivity from B. bas-siana or Metarhizium anisopliae, without direct proofof DNA damage, have purported that DNA damageand repair are responsible for these effects (Hunt etal 1994; Alves et al 1998; Braga et al 2001; Gilbertoet al 2001; Braga et al 2002; Gardner et al 1977; Ingliset al 1993, 1995; Inglis et al 1997; Morely-Davies et al1995; Costa et al 2001). Here we show direct proofthat it is UV-C irradiation of B. bassiana DNA thatcauses the formation of CPD and loss of viability(FIGS. 2 and 3).

Our experiments confirmed that T4 endonucleaseV analysis of UV-damaged DNA (Yasuda and Sekigu-chi 1970) is specific and accurate. The results of es-

625CHELICO ET AL: ULTRAVIOLET DAMAGE IN BEAUVERIA BASSIANA

FIG. 3. Relationship between UV dose, CPD/10 kb, and survival in B. bassiana conidiospores. A. Ethidium bromide-stained alkaline agarose gel of B. bassiana DNA isolated from conidiospores after exposure to varying doses of UV. Afterirradiation conidiospores were incubated with photoreactivating light for 6 h then DNA was isolated and digested with T4endonuclease V. Lanes: 1, DNA irradiated in vivo (120 J/m2) but not treated with T4 endonuclease V (control); 2, DNA notirradiated but treated with T4 endonuclease V (control, 0 J/m2); 39, 120, 240, 360, 480, 600, 720 and 1080 J/m2, respectively.B. Ethidium bromide-stained alkaline agarose gel of B. bassiana DNA isolated from conidiospores after exposure to varyingdoses of UV. After irradiation DNA was isolated from conidiospores immediately. Lanes: 1, DNA irradiated in vivo (120 J/m2) but not treated with T4 endonuclease V (control); 2, DNA not irradiated but treated with T4 endonuclease V (control,0 J/m2); 35, 120, 480 and 1200 J/m2, respectively. Lanes M are lHindIII/ fX174HaeIII markers with sizes (kb) of relevantbands shown in the margin. C. The number of CPD formed per 10 kb of DNA as a function of UV dose. The effect ofphotoreactivation (Phr) is assessed by comparison to no photoreactivation (control). D. Survival of B. bassiana conidiosporesafter incubation in photoreactivating conditions. The effect of photoreactivation (Phr) is assessed by comparison to nophotoreactivation (control). Each curve was constructed from the average of three independent experiments with barsreflecting the standard deviation of the mean.

timation of CPD in pAJ DNA (TABLE I) show thatvarying UV dose causes a discernable difference inthe number of CPD and their quantification is un-affected by the duration of enzymatic treatment. Sim-ilarly, varying DNA concentration of B. bassiana

showed that it had no limiting effect on CPD quan-tification.

Formation of CPD also was quantified from in vi-tro-irradiated B. bassiana DNA. It is known that CPDformation is more efficient between two adjacent thy-

626 MYCOLOGIA

mines than thymine-cytosine, cytosine-thymine or cy-tosine-cytosine (Ravanat et al 2001). The theoreticalmaximum number of CPD may never form in DNAdue to the sequence preference of CPD formation atthymine-thymine. Therefore, although the maximumCPD/10 kb we found for B. bassiana saturated at 28CPD/10 kb, it does not mean that all potential CPDsites in the DNA sequence were dimerized.

The maximum CPD formation in conidiosporeDNA of B. bassiana, is 15 per 10 kb and levels offafter ca. 480 J/m2 UV dose, which coincides with thetime when the maximum numbers of conidiosporeshave been killed by irradiation (FIG. 3C, D). Al-though (6-4)PP also form in DNA after UV-C irradi-ation these lesions cannot be quantified by alkalinegel electrophoresis. However, (6-4)PP lesions can bemended also by NER during repair incubation (Chel-ico et al 2003). The correlation (FIG. 3C, D) betweensurvival and CPD formation suggests that UV in-duced photoproducts in the DNA are a major causeof B. bassiana conidiospore killing. This is consistentwith other studies indicating CPD formation repre-sents 70% of the DNA damage induced by irradiation(Wang 1976).

The maximum number of CPD formed in B. bas-siana DNA irradiated in vivo if repair was preventedis 28 CPD/10 kb, and this occurred after 480 J/m2

of UV exposure (FIG. 3C). However the maximumnumber of CPD formed in B. bassiana DNA irradi-ated in vivo if allowed to repair CPD by photoreac-tivation was 15 CPD/10 kb and occurred after 480 J/m2 of UV exposure (FIG. 3C). This difference indi-cates a significant number of dimers formed in DNAirradiated in vivo are removed through their conver-sion to monomers by the repair system. The accu-mulated CPD in B. bassiana conidiospores levels offof after 480 J/m2 UV dose (FIG. 3C). This might bebecause the few surviving conidiospores still are ableto repair some DNA lesions and explains why thenumber CPD/10 kb does not continue to increasewhen the conidiospore viability has reached 0.003%(FIG. 3D).

Our study reports the first demonstration of num-ber of CPD quantified per 10 kb of UV-exposed EPFDNA. Although UV radiation was the first environ-mental constraint on EPF to be identified (Ignoffoet al 1977, Fargues 2003), no other quantitative stud-ies of DNA damage in EPF have been conducted. Wehave shown quantitatively that UV-C irradiation of B.bassiana causes formation of CPD and loss of conid-iospore viability. There can be three broader uses ofour findings on (i) timing of release of a fungal in-secticide (e.g. sunny or cloudy days), (ii) formula-tions that would mitigate UV induced DNA damage

(Cantrell et al 2001) and (iii) screening UV-tolerantisolates.

ACKNOWLEDGMENTS

A Natural Sciences and Engineering Research Council ofCanada Research Grant No. 493 (GG Khachatourians) andUniversity of Saskatchewan post-graduate scholarships to JLHaughian and L Chelico supported this research.

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