Problems utilizing an enzyme sensitive site assay for photorepair of ...

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Problems utilizing an enzyme sensitive site assay for photorepair of exogenous DNA with cell-free extracts made from amphibian embryos M. Alex Smith

Abstract: Attempts were made to assay the ultraviolet-B (UVB) damage repair ability of seven Ontario amphibian species using an enzyme sensitive site restriction-enzyme (ESS) assay. Cell-free protein extracts of amphibian eggs caused the degradation of even high (9 mg/mL) exogenous DNA concentrations. This type of signal loss is characteristic of nuclease digestion. High endogenous concentrations of amphibian nucleases appear to preclude the use of plasmid DNA–ESS assays to determine the UVB damage repair abilities of amphibian eggs. Proper estimation of amphibian ultraviolet damage repair characteristics, using any assay, is reliant upon the generation of cell-free protein extracts created from amphibian embryos covered in protective jelly. The process of releasing the embryo from the glycoprotein– carbohydrate jelly (“dejellying”) is achieved by shaking the jelly mass in a solution of 2% L-cysteine. Equivalent exposure to 2% L-cysteine results in a radically different end product with different amphibian taxa. These previously unreported phenomena have important implications for the production, standardization, and reporting of amphibian photorepair data. Résumé : Nous avons tenté d’évaluer la capacité de réparation des dommages causés par les ultra-violets-B (UVB) chez sept espèces d’amphibiens d’Ontario en utilisant un test ESS, test qui utilise une enzyme de restriction pour repérer les sites sensibles aux enzymes. Les extraits de protéines libres d’oeufs d’amphibiens ont entraîné la dégradation de concentrations exogènes d’ADN assez élevées (9 mg/mL). Ce type de perte de signal est caractéristique d’une digestion par nucléase. Des concentrations endogènes élevées de nucléases d’amphibien semblent rendre superflus les tests ADN–ESS à base de plasmides pour déterminer la capacité de réparation des dommages dus aux UVB des oeufs d’amphibien. Une estimation juste de cette capacité, par n’importe quel test, repose sur la génération d’extraits de protéines libres à partir d’embryons d’amphibiens recouverts d’une gelée protectrice. Le processus de libération de l’embryon de la gelée de glycoprotéine–hydrate de carbone consiste à agiter la masse gélatineuse dans une solution de L-cystéine 2 %. L’exposition à la L-cystéine 2 % aboutit à des produits totalement différents avec différents taxons. Ces phénomènes encore inédits influencent la production, la standardisation et la présentation de données sur la réparation des dommages causés par la lumière chez les amphibiens. [Traduit par la Rédaction]

Introduction

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When an organism is sessile (lacking behavioral means of ultraviolet-B (UVB) avoidance) or exists in habitats that tend to accentuate ultraviolet radiation exposure (Epel et al. 1999), it is important to estimate its ability to repair damage from UVB (280–320 nm) radiation. Most amphibian eggs meet the first criterion and, because of egg-deposition sites on the surface of the water column or in ephemeral water sources, many also meet the second criterion. Thus, a clear understanding of amphibian photorepair abilities affects our abilReceived November 19, 1999. Accepted June 19, 2000. M.A. Smith.1 Watershed Ecosystems Program, Trent University, Peterborough, ON K9J 7B8, Canada. 1

Present address: Redpath Museum, McGill University, Montréal, QC H3A 2K6, Canada (e-mail: [email protected]).

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ity to predict future population trends. Establishing quick nonradioactive methods of UVB damage repair ability will assist this process. This study was initiated to estimate the UVB damage repair ability of several Ontario amphibians, and the work reported here represents the initial evaluation of a method for determining UVB damage repair capacity. Legarski et al. (1987) demonstrated that the amount of UV damage per plasmid molecule could be determined through the topoisometric shift of plasmid conformation resolved in agarose-gel electrophoresis. Such a nonradioactive assay has been used (Dutta et al. 1993) to demonstrate the repair actions of photolyase. The enzyme sensitive site restrictionenzyme (ESS) assay utilized in this study depends on the recognition of the cyclobutane pyrimidine dimers (CBPDs) by the restriction enzyme T4 Endo V. T4 Endo V is a viral enzyme that specifically cuts the DNA strand at CBPDs, resulting in a change in conformation from closed covalent circles (form I or supercoiled) to relaxed molecules (form II or nicked circular; form III or linear) (Schrock and Lloyd © 2000 NRC Canada


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1993). The enzymatic activity of T4 Endo V has been measured by the rate of loss of form I DNA (Schrock and Lloyd 1993). Upon incubation with amphibian cell-free protein extracts (CFEs) and photoreactivating light (PRL), the dimer sites are removed and the proportion of form I as a percentage of the total DNA in each lane indicates photolyase activity. The conformation shift from form I to form II is evident through agarose-gel electrophoresis of the irradiated plasmid – amphibian protein extract subsequent to time under PRL. This enzyme system has been successfully used as a quick assay to determine the photolyase activity of several species of wild sea slugs (Carlini and Regan 1995). Photoenzymatic repair is likely a critical repair mechanism for amphibians. Although amphibian nucleotide excision repair has not been extensively quantified, in cases when it was (Freed et al. 1979), it was found not to play a significant role in amphibian DNA damage repair. However, it may be that the photolyase activity alone of some species will not be representative of their ability to repair UVB damage.

Methods Embryo collection and protein purification Methods of protein purification were adapted from Manly et al. (1980), Sancar et al. (1984), and Blaustein et al. (1994). Complete details regarding the egg-collection and protein-purification protocols are documented elsewhere,2 but are outlined here. The CFE protocols allow for optimum isolation of proteins in the photolyase size range. Briefly, 50 mL of egg replicates (frozen at –80°C upon collection) were warmed overnight from –80 to 4°C. A 10-mL volume of 2% L-cysteine was then added to each 50-mL container, and embryos were shaken at room temperature for between 1 and 3 h. L-cysteine acts as a dejellying agent by reducing disulphide bonds present in the jelly matrix. Shaking continued until the embryos were observed to be clustered at the bottom of the tube with no jelly surrounding and separating each embryo. Embryos were washed twice in cold phosphate buffered saline (PBS: 8 g NaCl, 0.2 g Na2HPO4, and 0.24 g KH2PO4 in 1 L of distilled H20, pH 7.4) and then centrifuged at 3000 rpm for 15 min in a KompSpin 21.50 rotor in a Beckman J2MC high-speed centrifuge. The supernatant was removed and the packed-cell volume (PCV) was estimated. Four times PCV of buffer I (10 mM Tris, 1 mM EDTA, and 5 mM DTT) was added and the mixture placed on ice for 20 min. Embryos were lysed and the resulting solution was then placed in a flask, where four times PCV of buffer II (50 mM Tris, 10 mM MgCl2, 2 mM DTT, 25% (w/v) sucrose, and 50% (v/v) glycerol) was added. One times PCV of saturated DNasefree ammonium sulfate was added. The solution was centrifuged at 20 000 rpm for 5 h at 4°C. The supernatant was decanted, 0.33 g of Sigma DNase-free ammonium sulfate was added per 1.0 mL of supernatant, while stirring, and 100 mL 1 N NaOH was added per 10 g of DNase-free ammonium sulfate. This solution was then centrifuged at 10 000 rpm for 30 min at 4°C. The supernatant was poured off; the resulting pellet was resuspended in 1/40th the measured supernatant volume of storage–dialysis buffer (25 mM Hepes, 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, and 16% (v/v) glycerol) and dialyzed overnight in tubing of molecular weight 14 000. The dialysate was centrifuged for 10 min at 10 000 rpm at 4°C. The supernatant was loaded onto a Sigma chromatographic column 2

Can. J. Zool. Vol. 78, 2000 with Blue Sepharose CL-6B, equilibrated with 0.1 M KCL and buffer B (50 mM Tris-HCl (pH 7.4), 1 mM EDTA, 10 mM 2mercaptoethanol, and 20% (v/v) glycerol), and washed with 0.1 M KCL and buffer B with 0.6 g/L ATP. The protein-rich fractions were collected, pooled, and dialyzed for 4–6 h against photolyase storage buffer (50 mM Tris (pH 7.4), 50 mM NaCl, 1 mM EDTA, 10 mM DTT, and 50% (v/v) glycerol). The resulting dialysate was collected and frozen at –20°C until used in the ESS assay. L-cysteine

dejellying results The amphibian embryos examined here displayed differential sensitivity to 2% L-cysteine as a dejellying agent. Ambystomid salamanders (Ambystoma laterale (blue-spotted salamander) and Ambystoma maculatum (yellow-spotted salamander)), Bufo americanus (the American toad), and the summer-laying ranids Rana clamitans (green frog) and Rana catesbieana (bullfrog) were susceptible to rapid embryo damage (Table 1). American toad and bullfrog embryo samples were damaged sufficiently to preclude their being included in studies reported elsewhere (Smith et al. 2000; Smith et al.2). Spring-laying ranids (Rana sylvatica (wood frog) and Rana pipiens (leopard frog)) were not as susceptible to the dejellying actions of 2% L-cysteine. Indeed, much longer exposure times to L-cysteine were required to remove embryos from jelly (Table 1). T4 Endo V ESS assay methods and results pBR322 plasmid DNA (Sigma) was prepared using the Bio-Rad Quantum Prep kit. Pyrimidine dimers were formed in the DNA by exposure to a photo-optic halogen bulb (1000W.120V.64743.Osram Corp.) at a distance of 10 cm for between 60 and 240 s (11.55–14.00 J/m2) in 16 mm polystyrene culture dishes at room temperature. A reaction mixture of CFE, photolyase assay buffer (50 mM Tris (pH 7.4), 50 mM NaCl, 1 mM EDTA, 10 mM DTT, and 50% (v/v) glycerol), and UVB-damaged plasmid was made and exposed to either PRL (10 cm from GE 15W Black Light lights) or darkness for 90 min. After light or dark treatment, the reaction mixture was incubated with T4 Endo V restriction endonuclease (Sigma) for 60 min at 37°C. After endonuclease incubation, 1–2 µL of 6× tracking dye (0.25% bromophenol blue and 40% (w/v) sucrose) was added to the mixture, and it was loaded onto a 1.0% agarose gel run in Tris-acetate – EDTA (TAE) buffer (0.04 M Tris-acetate and 0.001 M EDTA) containing 2–4 mL of 10 mg/mL ethidium bromide. Electrophoresis was carried out in the dark for 3–5 h at 20–45 V. The resulting banding patterns can be seen in Fig. 1. Gels were photographed and the positive print of each photograph was scanned using a digital scanner. The absorbance of any visible bands was analyzed by the National Institutes of Health Image package configured for the Windows platform (Scion Image), by comparing an unknown band with a series of known standards. The digital image was then used to quantify the absorbance of each band 1 from light and dark treatments. Photoreactivation of the plasmid– CFE complex allows for the monomerisation of pyrimidine dimers by photolyase in the CFE and, therefore, a reduced T4 Endo V digestion of the plasmid.

M.A. Smith, M. Berrill, and C.M. Kapron. Comparing photolyase activity and UVB jelly absorbance of several amphibian species from south-central Ontario. Submitted for publication. © 2000 NRC Canada



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Table 1. Differential effects of 2% L-cysteine as an agent for removing amphibian embryos from protective jelly. Species

Agitation time (min)

Protein concentration range (mg/mL)

Chorus frog Wood frog

1–2 60–120

1.37–1.45 1.51–7.99

Leopard frog

90–120

1.5–5.0

Blue-spotted salamander

0.5–20

0.13–0.49

Yellow-spotted salamander

0.5–20

0.03–1.86

American toad

10–60

0.09–0.23

Green frog

60–360

0.06–0.78

Bullfrog

7–120

None

Comments Little exposure or agitation required to remove embryos Little embryo damage after great agitation and L-cysteine exposure Little embryo damage after great agitation and L-cysteine exposure Rapid embryo destruction in L-cysteine, thus embryos dissected out of jelly capsule and briefly exposed to L-cysteine Rapid embryo destruction in L-cysteine, thus embryos dissected out of jelly capsule and briefly exposed to L-cysteine Complete embryo homogenization after L-cysteine exposure to remove embryos from jelly; not enough protein for photorepair analysis Greater exposure times required to remove embryos from jelly; some embryo degradation after L-cysteine exposure Embryos removed as they were freed from jelly; not enough protein for photorepair analysis

Note: For each species, the period of exposure to the dejellying agent is given. Also given is the protein concentration of the cell-free extract (CFE) that the embryos produced in the subsequent protein purification protocol, to represent a measure of the effects of 2% L-cysteine on the production of high-protein CFE. Other than the 2% L-cysteine treatment, the embryo CFE protocol was identical for each species. Qualitative observations are also included regarding the effects of using 2% L-cysteine on the embryo.

T4 Endo V digestion at CBPD sites results in a break in the DNA strand that is evident in electrophoresis as a conformational change from covalent closed circular molecules (form 1) to relaxed circular molecules (form II), (Fig. 1, lane 1). Since form I DNA fluoresces at 70% of the intensity of other conformations, the absorbance of this band was divided by 0.7 (Carlini and Regan 1995). The percentage of the DNA in form I conformation was determined by dividing the absorbance of form I (adjusted) by the sum of the absorbance of all three conformations and multiplying by 100. Subtracting the percentage of DNA in form I found after exposure to PRL from that found after no exposure to PRL allowed for an estimation of photorepair (Carlini and Reagan 1995). In treatments in which purified photolyase was incubated in the dark, dimers had not been repaired, T4 digestion was more complete, and therefore most of the DNA was in form II (Fig. 1). In the same treatment, but in the presence of 90 min of PRL, dimers were removed and, therefore, T4 digestion was less complete, and there was an increasing proportion of the total DNA in the form I conformation visible in lane 3 (Fig. 1). Lanes 4 and 5 (Fig. 1) were empty after incubation with leopard frog CFE. This result was characteristic for most exposures of DNA to amphibian CFE (21 of 24 assay attempts). Banding patterns were analyzed for gray tree frogs (Hyla versicolor), wood frogs, and yellow-spotted salamanders (data not shown). A time series for exposure to PRL displayed a negative relationship for the wood frog. As PRL is necessary for the catalysis of the CBPD, this trend is counterintuitive.

Discussion 2% L-cysteine as a dejellying agent Understanding amphibian ultraviolet repair abilities is of critical importance in maintaining a diverse functional ecosystem. Such an understanding is dependant on the accep-

tance that all amphibians are not anuran and that anurans do not represent all amphibians. In describing their process of dejellying to extract amphibian embryos, Blaustein et al. (1994) state that “Pro-eggs and eggs were first dejellied by treatment for 30–120 min with 2% cysteine….” (Blaustein et al. 1994, p. 1792, paragraph 2). This treatment, if applied consistently across the amphibian taxa collected in this study, would have destroyed the embryos of some species (bullfrogs and blue- and yellow-spotted salamanders), while for others (wood frog and leopard frog), half of the embryos in a jelly mass would not have been released (Table 1). It is critical that between-species differences in response to a methodological variable be reported. T4 Endo V ESS assay Hays et al. (1996, p. 450) state that “Levels of DNase in some amphibian egg extracts are high enough to cause significant degradation of exogenous DNA substrates.” Population declines, certainly not restricted to amphibian taxa, are occurring at an ever-increasing rate. Some of these declines are “normal” processes of population ebb and flow, but some are more closely related to anthropogenically induced stress. UVB represents such a stress that is increasing in areas that often do not appear to be experiencing changes in environmental stress. The speed at which these changes are occurring, and the fact that their effects will be felt all over the world, require that we have a quick inexpensive nonradioactive method of assessing one of the abilities organisms have to repair UVB damage. Currently, ESS assays have not filled this methodological need. Results reported elsewhere (see footnotes 2 and 3) support the declaration of Li et al. (1993, p. 4393) that, “One of the most sensitive assays for photolyase is the transformation assay.” The bacterial-transformation assay was much more efficient at illustrating dimer removal from exogenous DNA than the ESS assay completed here. In it, plasmid DNA was © 2000 NRC Canada



1872 Fig. 1. Agarose gel (1%) containing UV-irradiated pBR322 plasmid DNA. Lane 1 was irradiated with UVB radiation (11.55–14.00 J/m2). In addition to being given the same UVB exposure, the DNA in lanes 2 and 3 were mixed with purified bacterial photolyase (gift of Aziz Sancar) and then subsequently exposed to 90 min of photoreactivating light (lane 3) or 90 min of darkness (lane 2). Lanes 4 and 5 have undergone identical treatment, with the exception of the addition of leopard frog cellfree extract instead of purified photolyase. All lanes were incubated with T4 EndoV subsequent to exposure to UVB and the photoreactivating-light or darkness treatment. Exposing the reaction mixtures containing photolyase (lanes 3 and 5) to photoreactivating light results in the removal of CBPDs and, therefore, T4 digestion should be less complete; this results in an increasing proportion of DNA in the form I conformation. This is only evident in lane 3 and is discussed in the text.

Can. J. Zool. Vol. 78, 2000

assay is nearly as sensitive as the bacterial-transformation assay in quantifying the photolyase activity of amphibian CFE. Future analyses of these ESS assays with amphibian CFEs should attempt to estimate the concentrations of substratedamaging nucleases for a species before using the assay. This would allow a researcher to eliminate the potential use of the ESS assays a priori, if there were between-species differences in band digestion. Such differences need to be quantified. Between-species differences in response to methodological variables represent crucial information for reproducibility and must, therefore, be stringently reported.

Acknowledgements The author thanks Michael Berrill, Carolyn Kapron, and Michael Bidochka for laboratory space and advice. Aziz Sancar graciously provided advice and purified bacterial photolyase.

References

exposed to the same degrading agents that are present in amphibian CFE, but the assay was more sensitive to the presence of dimers and their subsequent removal via photorepair. Infrequent low intensity banding patterns characterized all ESS assays (see Smith et al. 2000 and footnote 2). Low absorbance plasmid DNA electrophoretic bands resulted subsequent to incubation with amphibian CFE and did not allow for proper estimation of the photolyase activity for the embryos. Results from assays using the H3 monoclonal antibody to detect dimer removal from exogenous DNA by amphibian protein (M.A. Smith, unpublished) suggest that this

Blaustein, A.R., Hoffman, P.D., Hokit, D.G., Kiesecker, J.M., Walls, S.C., and Hays, J.B. 1994. UV repair and resistance to solar UVB in amphibian eggs: a link to population declines? Proc. Natl. Acad. Sci. U.S.A. 91: 1791–1795. Carlini, D.B., and Regan, J.D. 1995. Photolyase activities of Elysia tuca, Busatell leachii and Haminaea antillarum (Mollusca: Opisthobranchia). J. Exp. Mar. Biol. Ecol. No. 189. pp. 219–232. Dutta, K., Hejmadi, W.S., and Verma, N.C. 1993. An assay for DNA photolyases using non-radioactive DNA and restriction enzymes. J. Photochem. Photobiol. B Biol. 18: 211–214. Epel, D., Hemela, K., Chick, M., and Patton, C. 1999. Development in the floating world: defenses of eggs and embryos against damage from UV radiation. Am. Zool. 39: 271–278. Freed, J.J., Hoess, R.H., Angelosanto, F.A., and Massey, H.C. 1979. Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells. Mutat. Res. 62: 235–239. Hays, J.B., Blaustein, A.R., Kiesecker, J.M., Hoffman, P.D., Pandelova, I., Coyle, D., and Richardson, T. 1996. Developmental responses of amphibians to solar and artificial UVB sources: a comparative study. Photochem. Photobiol. 64: 449–456. Li, Y.F., Kim, S.-T., and Sancar, A. 1993. Evidence for lack of DNA photoreactivating enzyme in humans. Proc. Natl. Acad. Sci. U.S.A. 90: 4389–4393. Legarski, R.J., Penkala, J.E., Peterson, C.A., and Wright, D.A. 1987. Repair of UV-induced lesions in Xenopus laevis oocytes. Mol. Cell. Biol. 7: 4317–4323. Sancar, A., Smith, F.W., and Sancar, G.B. 1984. Purification of Escherichia coli DNA photolyase. J. Biol. Chem. 259: 6028–6032. Schrock, R.D., and Lloyd, R.S. 1993. Site-directed mutagenesis of the NH2 terminus of T4 endonuclease V. The position of the alpha NH2 moiety affects catalytic activity. J. Biol. Chem. 268: 880–886. Smith, M.A., Kapron, C.M., and Berrill, M. 2000. Photolyase activity of wood frog (Rana sylvatica) embryo induced in vivo. Photochem. Photobiol. In press.

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