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Quantitation of supercoiled DNA cleavage in nonradioactive DNA: Application to lonizing radiation and synthetic endonuclease cleavage
ANALYTICAL
BIOCHEMISTRY
201,
80-86
(19%)
Quantitation of Supercoiled DNA Cleavage in Nonradioactive DNA: Application to Ionizing Radiation and Synthetic Endonuclease Cleavage’ Betsy M. Sutherland,
*12 Paula
V. Bennett,*
Kim
Conlon,?
Gary A. Epling,?
and John
C. Sutherland*
*Biology Department, Brookhaven National Laboratory Upton, New York 11973; and fChemz&y Department, University of Connecticut, Storrs, Connecticut 06268
Received
July
29, 1991
Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to “normalize” the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a proteinbased, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and nor-
1 Research supported by a grant from the Human Genome Project of the U.S. Department of Energy to B.M.S. and G.A.E., NIH Grants CA 23096 to B.M.S. and HG00371 to J.C.S., American Cancer Society Institutional Grant IRG-IN152C to G.A.E., and the Office of Health and Environmental Research of the U.S. Department of Energy. The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. * To whom correspondence should be addressed.
malizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays. 0 1992 A~demio
Press,
Inc.
Conversion of supercoiled DNA molecules to relaxed or linear forms is the basis for many biochemical assays. These topoisomers are easily separated on electrophoretie gels and visualized by fluorescence of bound ethidium bromide. However, quantitation of such reactions has been difficult because of the differential binding of ethidium to supercoiled DNA vs the other forms (1) and the nonlinear response of photographic film (2-4). These difficulties led some investigators (e.g., Ref. (5)] to obtain only qualitative information from their supercoil conversion data. Other investigators prepared radioactive DNA, and, after electrophoresis and location of the bands by ethidium fluorescence, cut out the bands and determined the quantity of radioactivity in each fraction. Some investigators determined R, the relative binding of ethidium to relaxed compared to supercoiled DNAs, under their experimental conditions (6-9) and used the resulting values to compute the quantity of each topoisomer. Other investigators used an R value from the literature, assuming that R is a constant. However, the range of R values obtained by different investigators indicates that the ratio of ethidium binding to the different species varies markedly with experimental conditions. Bauer and Vinograd (1) estimated that at high ethidium concentrations only about 65% as much ethidium bound to supercoiled SV40 as did to the re-
80 All
Copyright 0 1992 rights of reproduction
0003-269’7/92 $3.00 by Academic Press, Inc. in any form reserved.
ELECTROPHORETIC
QUANTITATION
laxed form (assuming that the quantum yields of fluorescence for ethidium bound to supercoiled and nonsupercoiled molecules are the same; this corresponds to R = 1.54). Ciulla et al. (8) obtained a value for R of 1.66; Lloyd et al. (6), 1.44; and Roots et al. (7), 1.25. Masnyk and Minton (9) found that the “correction factor for reduced binding of ethidium bromide by Form I DNA was negligible,” suggesting a binding ratio of 1. Since the variation in the published values of R is large, the use of values determined by others under different gel, buffer, or staining conditions may lead to significant errors in the quantitation of the fraction of DNA in each topoisomer. One solution to the problem would be to load exactly equal quantities of DNA in each gel lane and to quantitate the absolute amount of supercoiled DNA remaining after the various treatments. Quantitative loading of samples, especially into submerged agarose gels, is difficult, time consuming, and subject to inaccuracies. We have developed a method in which an electrophoretically distinct linear DNA is added as a mass standard or “normalizing” DNA; this addition circumvents errors resulting from uncertainties in R and also eliminates the need for quantitative sample loading. To evaluate the utility of this approach, we evaluated three methods for quantitating the conversion of supercoiled DNA to other forms: first, quantitative loading of samples onto a gel and determining the absolute quantities of supercoiled DNA in each sample; second, including a normalizing DNA at a fixed ratio to the supercoiled DNA before samples are partitioned for the conversion reaction and determining the relative quantities of supercoiled to normalizing DNA in each sample; and third, adding the normalizing DNA to each sample after the conversion reaction and determining the relative quantities of supercoiled and normalizing DNAs. The second method, addition of a normalizing molecule with the substrate DNA before partitioning for the conversion reaction, gives the best reproducibility and avoids the requirement of quantitative sample loading on electrophoretic gels.
MATERIALS
AND
METHODS
DNAs Supercoiled PET-2 (10) and pUC 18 DNAs were prepared by detergent lysis, heat treatment, centrifugation, and polyethylene glycol precipitation, followed by extraction with chloroform-isoamyl alcohol, phenol, and chloroform. Plasmid pUC 18 was treated with BglI (Boehringer-Mannheim) using the manufacturer’s conditions. The restriction digestion was terminated by the addition of EDTA to 10 mM and heating to 65°C for 20 min. The DNA was dialyzed into 10 mM NaPO, buffer, pH 7.2.
OF
SUPERCOILED
DNA
81
CLEAVAGE
Irradiations Supercoiled PET-2 DNA was irradiated with 0 to loGy 137Csy rays as follows: Solution A containing PET-2 DNA at 10 ng/pl was irradiated in 10 mM NaPO, buffer, pH 7.2; Solution B contained 5 ng/pl PET-2 DNA and 5 ng/pl BglI-treated pUC 18 in the same buffer and was used in Treatment 2. After irradiation, Solution A was divided; to one part (Treatment 3), BglI-digested pUC 18 was added to each sample to yield a final concentration of each DNA of 5 ng/pl, while to the other (Treatment l), 10 mM NaPO, buffer, pH 7.2, was added to each sample to give a final PET-2 concentration of 5 ng/pl. A gel loading mixture of 0.125% bromophenol blue in 50% glycerol was added to each sample before electrophoresis.
Synthetic
Endonuclease
Labeling
and Cleavage
T7 RNA polymerase was labeled with an average of 17 rose bengals/protein by a procedure that will be described in detail elsewhere. The labeled polymerase was added to a DNA mix containing (for the detection of promoter-specific cleavage) 21 ng/pl supercoiled PET-2 DNA (which contains a T7 promoter) and 21 ng/pl BgZI-restricted pUC 18 in 20 mM NaPO,, pH 7.7,50 mM NaCl, 5% glycerol, 1 mM dithiothreitol, 5 mM GTP, 10 mM MgCl,, and 0.1 mg/ml gelatin or (for the detection of nonspecific cleavage) supercoiled pUC 18 and BgZIrestricted pUC 18, both at 21 ng/pl, in the same buffer. Each sample was divided into two aliquots; all samples were incubated for 15 min at 37°C with argon bubbling, and then one aliquot was exposed to cool white fluorescent light at 37°C for 30 min while a companion aliquot was kept in the dark at 37°C for 30 min. A neutral stop mixture containing 0.125% bromophenol blue, 0.05% sodium dodecyl sulfate (SDS)3 in 50% glycerol was added to each sample before electrophoresis. Siliconized micropipetter tips were used in the application of samples to the agarose gels.
Electrophoresis
and DNA Analysis
DNAs were electrophoresed in 1% agarose (Sigma Type II) gels in 40 mM Tris, 20 mM acetate, 20 mM EDTA, 0.18 M NaCl, pH 8. The molten agarose had been filtered through a 0.8 pm filter to remove fluorescent particles. Horizontal agarose gels were electrophoresed in a Bio-Rad Mini-Sub Cell for 1.5 h at 37 V in Tris-acetate buffer. After electrophoresis, the gel was stained for 10 min in ethidium bromide (1 pg/ml in water) and destained for 40 min in water, and a quantitative electronic image of the ethidium fluorescence on the gel was obtained using either the charge-coupled
3 Abbreviations used: CCD, charge-coupled strand break; SDS, sodium dodecyl sulfate.
device;
ssb,
single-
82
SUTHERLAND
device (CCD)-based imaging system described by Sutherland et al. (11) or a “second-generation” CCD-based system that will be described elsewhere. The quantity of ethidium fluorescence corresponding to DNA bands of interest was determined in each gel lane and #, the fraction of supercoiled DNA, calculated. RESULTS AND DISCUSSION Evaluation of methods for the conversion of supercoiled DNA to its relaxed form requires a simple method of inducing single-strand breaks in DNA. Ionizing radiation induces single-strand breaks in DNA in a single-hit reaction, and thus the fraction of the initial supercoiled molecules that have not experienced a single-strand break (ssb) after exposure to a dose D is given by the equation 4(D) = exp-“n),
s.0
AL.
PET-2 decreases upon y irradiation, with a corresponding increase in the relaxed form. At the highest doses, a small amount of linear DNA appears at a position of slightly greater mobility than that of the relaxed form. The linear molecules result either from a double-strand break or two closely opposed single-strand breaks. The linear normalizing pUC 18 DNA, however, remains at approximately the same concentration in all the lanes, with differences reflecting variations only in sample application in the lanes. Figure 1B shows the results of the quantitation of the supercoil conversion in the gel shown in Fig. 2. The total fluorescence from supercoiled PET-2 DNA and from pUC 18 DNA was determined for each sample and the ratio of supercoiled PET-2 DNA to pUC 18 determined. The fraction of supercoiled DNA in each irradiated sample, 4, was then calculated from the equation.
VI
where ~(0) is the mean number of single-strand breaks per molecule resulting from dose D of radiation. If p(D) is a linear function of the dose, we can write p (D) = ~‘a D. Thus, when lag(4) is plotted as a function of dose, we expect a straight line with slope -P’. Figure 1 shows the results of three methods of analysis of such a conversion. Figure 1A shows the results of irradiation of the supercoiled DNA alone, quantitative loading of each irradiated sample, electrophoresis, ethidium staining, and calculation of the area of the supercoiled band in each sample lane (Treatment 1). The fraction of supercoiled DNA, 4, in each sample is calculated from the equation (b(D) = +,
ET
PI
where Fsp is the integrated fluorescence of the supercoiled band at dose D, and Fs,o is the integrated fluorescence of the supercoiled band in the unirradiated sample. The open and closed symbols in Fig. 1 represent results from two independent gels. The results of statistical analysis of the data are shown in Table 1. The data show increasing fluctuations at the higher doses; as the area of the supercoiled band decreases, small variations in lane-to-lane gel loading become increasingly significant. Moreover, accurate quantitative loading of a horizontal agarose gel submerged under buffer is tedious and time consuming. We next evaluated the addition of a normalizing DNA to the supercoiled DNA stock before subdivision of the stock for the various doses of y rays (Treatment 2). Figure 2 shows an electronic image of fluorescence from an ethidium bromide-stained gel containing supercoiled PET-2 DNA as a test DNA and BglI-restricted pUC 18 as a normalizing DNA. The quantity of supercoiled
where Fsp and Fs,o are as defined previously and FNp and FN,O are the integrated fluorescence intensities of the bands of normalizing pUC 18 DNA that received doses D and 0, respectively. The use of the normalizing DNA allows correction for lane-to-lane variation in sample application. Both gels (0 and +) gave similar results; a statistical analysis of the data is shown in Table 1. Although addition of the normalizing DNA before division of the supercoiled stock gave better results than analysis of the test DNA alone, in some experimental protocols, such an additional DNA might interfere with the reaction being studied. We thus tested whether the standardizing DNA could be added to each sample after the conversion reaction (Treatment 3). This would avoid the presence of a normalizing DNA in the supercoil cleavage reaction, but offer the advantage of its presence during quantitation. It should facilitate gel loading, but would be influenced by differences in pipetting the normalizing DNA into each sample, small volume changes during handling and irradiation, etc. Figure 1C shows that this method gave good agreement between the two independent gels, but was less accurate at low levels of conversion of supercoils (i.e., low doses). The three methods can be compared using the data shown in Table 1. First, the slopes of the regression lines are similar within the 95% confidence limit (-2~). However, Treatment 2 has the highest correlation coefficient, lowest standard deviation, and residual variance. In cases in which the addition of a standardizing plasmid during supercoil cleavage is undesirable, Treatment 3 gives reasonable standard deviation and residual variance, but does show significant scatter at low levels of supercoil conversion (See Fig. 1C). Treatment 1, in which no standardizing plasmid is used, gives a higher
ELECTROPHORETIC
QUANTITATION
OF
SUPERCOILED
DNA
CLEAVAGE
0.1 0
2
4
6
Dose
(Gy)
8
10
0
4
6
Dose
(Gy)
2
4
6
DOSE
(Gy)
8
10
10
FIG. 1. Quantitation of the conversion of supercoiled DNA to its relaxed form by three methods. (A) SupercoiledpET-2 DNA was irradiated in solution; equal aliquots of each sample were applied quantitatively to an agarose gel (Treatment 1). After electrophoresis, staining with ethidium, and destaining, an electronic image was obtained. The quantity of supercoiled DNA was determined for each sample from the integrated lane profile. The symbols (A and A) show results obtained in two independent gels. (B) Supercoiled PET-2 DNA was mixed with linear pUC 18 DNA as an internal standard or “normalizing” molecule and the solution was then divided into samples for each irradiation treatment; samples were applied to agarose gels, electrophoresed, and stained and a quantitative image was obtained (Treatment 2). The fluorescence from supercoiled PET-2 DNA and from pUC 18 DNA was determined for each sample from the integrated lane profile and the ratio of supercoiled PET-2 DNA to pUC 18 determined. The fraction of DNA that remained supercoiled at each dose was then obtained by dividing the ratio of supercoiled DNA to normalizing DNA at each dose by the value of that ratio in the unirradiated sample, as indicated in Eq. [3]. Results for two gels are shown (0, +). (C) Supercoiled PET-2 DNA was irradiated, and, after irradiation, linear pUC-18 was added to each sample separately (Treatment 3). The samples were electrophoresed, the DNA was stained with ethidium, and a quantitative image was obtained. The fraction of supercoiled DNA was calculated as in (B). The results of two independent gels are shown (0, 0).
standard deviation and residual variance, but gives a rate of cleavage that is similar to that obtained by the other methods. Quantitative loading of submerged agarose gels, however, is tedious and time consuming.
We used a variation of Treatment 2, which is described below, to quantitate the nicking of supercoiled DNA in a more complex system: cleavage of supercoiled DNA containing a T7 promoter site by the T7 RNA
84
SUTHERLAND
ET
TABLE Statistical Conditions DNA
Treatment
(standardizing addition) None Before After
1 2 3 a Standard
Comparison
deviation
of the slope
of Three ri (ssb/molecule/Gy) -0.081 -0.085 -0.079
of the dose-response
AL.
1
Methods
of Supercoil
Quantitation
$1 (ssb/molecule/Gy) 0.0261 0.0068 0.0141
Residual variance 0.0674 0.0046 0.0196
Correlation coefficient 0.975 0.997 0.992
function.
polymerase-based synthetic photoendonuclease. We chose pUC 18 as a normalizing DNA, as it does not contain a specific binding site for the polymerase. The polymerase does not bind to pUC 18 at the lower ratios of polymerase to DNA used in these experiments. T7 RNA polymerase was derivatized with an average of 17 rose bengal molecules per protein and incubated in the presence of supercoiled PET-2 DNA, which contains a strong promoter site for the polymerase, and EcoRIdigested pUC 18 as normalizing DNA. After preincubation, the samples were divided; one part of each sample was exposed to visible light (activating the rose bengal and initiating DNA cleavage), while the rest of each sample was kept in the dark. Samples were mixed with a neutral gel loading mixture containing SDS and then electrophoresed on neutral gels. After ethidium staining and destaining, a digital image was obtained using a CCD camera, and the areas of the relaxed (see below) and normalizing DNAs in each lane were determined. The fraction of supercoiled DNA converted to relaxed DNA in the light vs the fraction converted in the dark (due to non-light-dependent nicking) was calculated for each sample. Figure 3 shows that the synthetic endonuclease cleaves the DNA in samples exposed to visible light, with little DNA nicking in the absence of light exposure. Two factors complicate the analysis of supercoil nicking: first, the protein or light might nick the DNA nonspecifically, independently of their concerted endonucleolytic activity. Thus appropriate dark and light controls are required at each protein concentration. Second, measurement of initial cleavage rates requires quantitation at low levels of conversion and would thus be facilitated by measurement of the increase in relaxed molecules (a large change in an initially small number, rather than a small change in the large area of the supercoil band). We can calculate the fraction of the supercoiled molecules remaining and hence the average number of single-strand breaks per molecule induced by a certain treatment from the fluorescence intensities of only the relaxed molecules, provided that we also know the normalized intensity of the fluorescence of all of the experimental DNA-the PET-2 in our experimentswhen none of it is supercoiled. We denote this normal-
ized fluorescence intensity as FE,T/FN,T, where the “T” denotes “total” DNA in the sample and, as above, the “E” denotes “experimental” and the “N” denotes “normalizing.” The value of FE,T/FN,T can be determined in several different ways. If the value of R is known for the given experimental conditions, the value of FE,TIFN,T can be obtained from the equation F E.T
R-F,, F NT =L+F’F NJ
FRD ND
where, for the low doses that are of interest, we assume that there will be no significant number of linear molecules formed.4 Another possibility is that the ratio of the total experimental DNA to the normalizing DNA is known from the design of the experiment. A third possibility is to measure FE,-JFN,T by including in the gel a sample of the DNA mixture that has been treated with a restriction endonuclease that linearizes all the supercoiled and relaxed DNA. Combining Eqs. [3] and [4] results in the desired expression
PI If the number of breaks per molecule is linearly proportional to the extent of the treatment, a plot of the logarithm of the expression on the right-hand side of Eq. [5] will result in a straight line, the negative slope of which is the number of strand breaks per molecule per unit “dose,” where dose is used in the more general sense of “treatment.” Figure 3 shows the resulting data for cleavage by the synthetic endonuclease derivatized with 17 rose bengals per polymerase molecule. In samples exposed to 30 min visible light, the DNA was cleaved by the polymerase, with the extent of nicking dependent on the quantity of labeled polymerase added. In the absence of light, the ’ Generalizing Eq. [4] to allow for the production of linear molecules is straitforward provided that the binding and quantum yield of fluorescence of ethidium bromide to nicked circular and linear DNAs are identical.
ELECTROPHORETIC
QUANTITATION
FIG. 2. Electronic image of an electrophoretic gel showing cleavage of supercoiled PET-2 DNA by ionizing radiation. The direction of electrophoresis is from top to bottom; the bands, in order from top to bottom, are relaxed circles, linear molecules (if present), supercoiled molecules, and two BglI fragments of pUC 18, the larger of which was used as an internal standard. The supercoiled PET-2 DNA and the pUC 18 internal standard DNA were mixed, and samples were exposed to 0,0.5,1,2,4,6,8,8, and 10 Gy of 137Cs y rays, shown from left to right. The depletion of supercoiled DNA and increase in relaxed molecules contrast with the approximately constant quantities of standardizing DNA.
polymerase does not cleave the DNA. DNA without a promoter site is not cleaved significantly in the light compared to that cleaved in the dark control. It is important to consider the criteria for the selection of a normalizing DNA. First, the normalizing DNA must be easily separable from the test system in the gel system used for analysis of the test DNA. Second, the normalizing DNA must be easy to quantitate. Third, it must not interfere with the supercoil conversion reaction, nor be altered by the reaction so as to interfere with cleavage, separation, or subsequent quantitation. In the in vitro irradiations, normalizing DNA could be added to the test supercoiled DNA without consideration of its sequence. Linear pUC 18 is easily separated from supercoiled PET-2, and because of both its small size and its topology, it is less susceptible per molecule to observable effects of strand breakage. For reactions mediated by sequence-specific DNA binding proteins (such as the polymerase-based synthetic endonuclease), the normalizing DNA must not contain binding sites for the enzyme observable under the experimental reaction or gel conditions. The use of the normalizing plasmid allows facile quantitation of DNA cleavage, even at low levels of conversion. The use of a normalizing DNA can be applied to many other reactions. For example, we have used it in quantitating pyrimidine dimer formation, measured by specific nicking at dimer sites by the dimer-specific uv endonuclease from A4icrococcus luteus (12). In this case, the substrate DNA was uv irradiated, while the normalizing DNA was unirradiated. We have also used this approach to measure repair of dimers by the 40-kDa photoreactivating enzyme from Escherichia coli (13) in a
OF
SUPERCOILED
DNA
85
CLEAVAGE
coupled reaction, using uv-irradiated substrate DNA and unirradiated normalizing DNA, followed by treatment with the M. luteus uv endonuclease. The normalizing DNA method also greatly facilitates the quantitation of DNA retardation assays for measuring the binding of proteins to specific DNA sites (14). In this case, the substrate DNA contains the binding site for the protein of interest, while the normalizing DNA lacks the site. After incubation, the samples are loaded onto a neutral gel compatible with retention of DNAprotein binding and electrophoresed, and the gel is stained and destained as usual. The areas of the free (unbound) substrate DNA and the normalizing DNA are determined for each lane, and the ratio of free DNA/ normalizing DNA is determined. This method allows rapid loading of the gel (which is difficult for methods requiring quantitative transfer of gel samples) and thus maximizes the detection of labile DNA-protein complexes. We have used electronic imaging for the quantitation of fluorescence of DNA-bound ethidium because of its accuracy of DNA measurement (ll), short exposure times (minimizingethidiumphotobleaching), andconvenience. However, similar information can be obtained using photographic film, with attention to standard methods of compensating for film nonlinearity [e.g., Refs. (2-4, 15)] and failure of time-intensity reciprocity [e.g., Ref. (IS)]. Although the addition of the normalizing DNA before
E 2
4 : ;=:
1
_---
z----*
_____
---
----a i
Iv
0
4
6
12
I 16
RB-RNAP/DNA
FIG. 3. T7 RNA polymerase labeled with 17 rose bengal molecules/ protein was added to a mixture of supercoiled PET-2 DNA, which contains a T7 promoter, and BglI-linearized pUC 18, which has no polymerase binding site. The samples were preincubated with argon bubbling and then part of each sample was exposed for 30 min to visible light (O), while a companion part of each sample was kept in the dark (A). Samples were electrophoresed on agarose gels and stained with ethidium, and an electronic image was obtained, the fluorescent intensities of the relaxed and normalizing DNA bands were determined. The fraction of supercoiled DNA remaining was calculated from these intensities using Eq. [5].
86
SUTHERLAND
the partitioning of the stock solution is clearly the method of choice when possible, some reactions may not be compatible with the addition of normalizing DNAs. The data shown in Fig. 1 as well as the results of additional experiments (data not shown) allow the evaluation of the other methods that we tested. First, all three methods gave similar slopes. The residual variances in Treatments 3 (normalizing plasmid added after the conversion reaction) and 2 (normalizing plasmid added before the division of DNA stock) were similar, but the variance in Treatment 1 (no normalizing DNA) was significantly higher than that in the other two methods. Thus the method of second choice is Treatment 3, in which the reaction converting supercoils to other forms is carried out first, and then a normalizing DNA is added before gel loading. The higher variance and technical difficulty of Treatment 1 (no normalizing DNA) make it the third choice. It is clear that the use of the normalizing plasmid is the preferred method and can be applied to many reactions.
We thank Dr. Giovanni Ciarrocchi for useful Thompson for statistical analyses, and Anne Katz desi for assistance.
discussions, and Matthew
Keith Ran-
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5. Wang, T. P., Kagan, Photo&m. Photobiol. 6. Lloyd, istry
R. S., Haidle,
J., Tuveson, 53,463-467.
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(Grossman, L., and Academic Press, San
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C. W., and Robberson,
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11. Sutherland, J. C., Lin, B.-H., Monteleone, D. C., Mugavero, J. H., Sutherland, B. M., and Trunk, J. (1987) Anal. Biochem. 163, 446-456. 12. Carrier, W. L., and Setlow, R. B. (1970) J. Bacterid 102, 173186. 13.
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2. Pulleyblank, D. E., Shure, Acids Res. 4,1409-1418.
ET
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G., Brash, D. E., P. C. (1986) Bio-
14. Sutherland, J. C., Sutherland, B. M., Emrick, A., Monteleone, D. C., Ribeiro, E. A., Trunk, J., Son, M., Serwer, P., Poddar, S. K., and Maniloff, J. (1991) BioTechniques 10,492-497. 15. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Biochem. 56,335341. 16. Ribeiro, them.
E. A., Larcom,
181,197-208.
L. L., and Miller,
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And.
Bio-
BIOCHEMISTRY
201,
80-86
(19%)
Quantitation of Supercoiled DNA Cleavage in Nonradioactive DNA: Application to Ionizing Radiation and Synthetic Endonuclease Cleavage’ Betsy M. Sutherland,
*12 Paula
V. Bennett,*
Kim
Conlon,?
Gary A. Epling,?
and John
C. Sutherland*
*Biology Department, Brookhaven National Laboratory Upton, New York 11973; and fChemz&y Department, University of Connecticut, Storrs, Connecticut 06268
Received
July
29, 1991
Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to “normalize” the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a proteinbased, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and nor-
1 Research supported by a grant from the Human Genome Project of the U.S. Department of Energy to B.M.S. and G.A.E., NIH Grants CA 23096 to B.M.S. and HG00371 to J.C.S., American Cancer Society Institutional Grant IRG-IN152C to G.A.E., and the Office of Health and Environmental Research of the U.S. Department of Energy. The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. * To whom correspondence should be addressed.
malizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays. 0 1992 A~demio
Press,
Inc.
Conversion of supercoiled DNA molecules to relaxed or linear forms is the basis for many biochemical assays. These topoisomers are easily separated on electrophoretie gels and visualized by fluorescence of bound ethidium bromide. However, quantitation of such reactions has been difficult because of the differential binding of ethidium to supercoiled DNA vs the other forms (1) and the nonlinear response of photographic film (2-4). These difficulties led some investigators (e.g., Ref. (5)] to obtain only qualitative information from their supercoil conversion data. Other investigators prepared radioactive DNA, and, after electrophoresis and location of the bands by ethidium fluorescence, cut out the bands and determined the quantity of radioactivity in each fraction. Some investigators determined R, the relative binding of ethidium to relaxed compared to supercoiled DNAs, under their experimental conditions (6-9) and used the resulting values to compute the quantity of each topoisomer. Other investigators used an R value from the literature, assuming that R is a constant. However, the range of R values obtained by different investigators indicates that the ratio of ethidium binding to the different species varies markedly with experimental conditions. Bauer and Vinograd (1) estimated that at high ethidium concentrations only about 65% as much ethidium bound to supercoiled SV40 as did to the re-
80 All
Copyright 0 1992 rights of reproduction
0003-269’7/92 $3.00 by Academic Press, Inc. in any form reserved.
ELECTROPHORETIC
QUANTITATION
laxed form (assuming that the quantum yields of fluorescence for ethidium bound to supercoiled and nonsupercoiled molecules are the same; this corresponds to R = 1.54). Ciulla et al. (8) obtained a value for R of 1.66; Lloyd et al. (6), 1.44; and Roots et al. (7), 1.25. Masnyk and Minton (9) found that the “correction factor for reduced binding of ethidium bromide by Form I DNA was negligible,” suggesting a binding ratio of 1. Since the variation in the published values of R is large, the use of values determined by others under different gel, buffer, or staining conditions may lead to significant errors in the quantitation of the fraction of DNA in each topoisomer. One solution to the problem would be to load exactly equal quantities of DNA in each gel lane and to quantitate the absolute amount of supercoiled DNA remaining after the various treatments. Quantitative loading of samples, especially into submerged agarose gels, is difficult, time consuming, and subject to inaccuracies. We have developed a method in which an electrophoretically distinct linear DNA is added as a mass standard or “normalizing” DNA; this addition circumvents errors resulting from uncertainties in R and also eliminates the need for quantitative sample loading. To evaluate the utility of this approach, we evaluated three methods for quantitating the conversion of supercoiled DNA to other forms: first, quantitative loading of samples onto a gel and determining the absolute quantities of supercoiled DNA in each sample; second, including a normalizing DNA at a fixed ratio to the supercoiled DNA before samples are partitioned for the conversion reaction and determining the relative quantities of supercoiled to normalizing DNA in each sample; and third, adding the normalizing DNA to each sample after the conversion reaction and determining the relative quantities of supercoiled and normalizing DNAs. The second method, addition of a normalizing molecule with the substrate DNA before partitioning for the conversion reaction, gives the best reproducibility and avoids the requirement of quantitative sample loading on electrophoretic gels.
MATERIALS
AND
METHODS
DNAs Supercoiled PET-2 (10) and pUC 18 DNAs were prepared by detergent lysis, heat treatment, centrifugation, and polyethylene glycol precipitation, followed by extraction with chloroform-isoamyl alcohol, phenol, and chloroform. Plasmid pUC 18 was treated with BglI (Boehringer-Mannheim) using the manufacturer’s conditions. The restriction digestion was terminated by the addition of EDTA to 10 mM and heating to 65°C for 20 min. The DNA was dialyzed into 10 mM NaPO, buffer, pH 7.2.
OF
SUPERCOILED
DNA
81
CLEAVAGE
Irradiations Supercoiled PET-2 DNA was irradiated with 0 to loGy 137Csy rays as follows: Solution A containing PET-2 DNA at 10 ng/pl was irradiated in 10 mM NaPO, buffer, pH 7.2; Solution B contained 5 ng/pl PET-2 DNA and 5 ng/pl BglI-treated pUC 18 in the same buffer and was used in Treatment 2. After irradiation, Solution A was divided; to one part (Treatment 3), BglI-digested pUC 18 was added to each sample to yield a final concentration of each DNA of 5 ng/pl, while to the other (Treatment l), 10 mM NaPO, buffer, pH 7.2, was added to each sample to give a final PET-2 concentration of 5 ng/pl. A gel loading mixture of 0.125% bromophenol blue in 50% glycerol was added to each sample before electrophoresis.
Synthetic
Endonuclease
Labeling
and Cleavage
T7 RNA polymerase was labeled with an average of 17 rose bengals/protein by a procedure that will be described in detail elsewhere. The labeled polymerase was added to a DNA mix containing (for the detection of promoter-specific cleavage) 21 ng/pl supercoiled PET-2 DNA (which contains a T7 promoter) and 21 ng/pl BgZI-restricted pUC 18 in 20 mM NaPO,, pH 7.7,50 mM NaCl, 5% glycerol, 1 mM dithiothreitol, 5 mM GTP, 10 mM MgCl,, and 0.1 mg/ml gelatin or (for the detection of nonspecific cleavage) supercoiled pUC 18 and BgZIrestricted pUC 18, both at 21 ng/pl, in the same buffer. Each sample was divided into two aliquots; all samples were incubated for 15 min at 37°C with argon bubbling, and then one aliquot was exposed to cool white fluorescent light at 37°C for 30 min while a companion aliquot was kept in the dark at 37°C for 30 min. A neutral stop mixture containing 0.125% bromophenol blue, 0.05% sodium dodecyl sulfate (SDS)3 in 50% glycerol was added to each sample before electrophoresis. Siliconized micropipetter tips were used in the application of samples to the agarose gels.
Electrophoresis
and DNA Analysis
DNAs were electrophoresed in 1% agarose (Sigma Type II) gels in 40 mM Tris, 20 mM acetate, 20 mM EDTA, 0.18 M NaCl, pH 8. The molten agarose had been filtered through a 0.8 pm filter to remove fluorescent particles. Horizontal agarose gels were electrophoresed in a Bio-Rad Mini-Sub Cell for 1.5 h at 37 V in Tris-acetate buffer. After electrophoresis, the gel was stained for 10 min in ethidium bromide (1 pg/ml in water) and destained for 40 min in water, and a quantitative electronic image of the ethidium fluorescence on the gel was obtained using either the charge-coupled
3 Abbreviations used: CCD, charge-coupled strand break; SDS, sodium dodecyl sulfate.
device;
ssb,
single-
82
SUTHERLAND
device (CCD)-based imaging system described by Sutherland et al. (11) or a “second-generation” CCD-based system that will be described elsewhere. The quantity of ethidium fluorescence corresponding to DNA bands of interest was determined in each gel lane and #, the fraction of supercoiled DNA, calculated. RESULTS AND DISCUSSION Evaluation of methods for the conversion of supercoiled DNA to its relaxed form requires a simple method of inducing single-strand breaks in DNA. Ionizing radiation induces single-strand breaks in DNA in a single-hit reaction, and thus the fraction of the initial supercoiled molecules that have not experienced a single-strand break (ssb) after exposure to a dose D is given by the equation 4(D) = exp-“n),
s.0
AL.
PET-2 decreases upon y irradiation, with a corresponding increase in the relaxed form. At the highest doses, a small amount of linear DNA appears at a position of slightly greater mobility than that of the relaxed form. The linear molecules result either from a double-strand break or two closely opposed single-strand breaks. The linear normalizing pUC 18 DNA, however, remains at approximately the same concentration in all the lanes, with differences reflecting variations only in sample application in the lanes. Figure 1B shows the results of the quantitation of the supercoil conversion in the gel shown in Fig. 2. The total fluorescence from supercoiled PET-2 DNA and from pUC 18 DNA was determined for each sample and the ratio of supercoiled PET-2 DNA to pUC 18 determined. The fraction of supercoiled DNA in each irradiated sample, 4, was then calculated from the equation.
VI
where ~(0) is the mean number of single-strand breaks per molecule resulting from dose D of radiation. If p(D) is a linear function of the dose, we can write p (D) = ~‘a D. Thus, when lag(4) is plotted as a function of dose, we expect a straight line with slope -P’. Figure 1 shows the results of three methods of analysis of such a conversion. Figure 1A shows the results of irradiation of the supercoiled DNA alone, quantitative loading of each irradiated sample, electrophoresis, ethidium staining, and calculation of the area of the supercoiled band in each sample lane (Treatment 1). The fraction of supercoiled DNA, 4, in each sample is calculated from the equation (b(D) = +,
ET
PI
where Fsp is the integrated fluorescence of the supercoiled band at dose D, and Fs,o is the integrated fluorescence of the supercoiled band in the unirradiated sample. The open and closed symbols in Fig. 1 represent results from two independent gels. The results of statistical analysis of the data are shown in Table 1. The data show increasing fluctuations at the higher doses; as the area of the supercoiled band decreases, small variations in lane-to-lane gel loading become increasingly significant. Moreover, accurate quantitative loading of a horizontal agarose gel submerged under buffer is tedious and time consuming. We next evaluated the addition of a normalizing DNA to the supercoiled DNA stock before subdivision of the stock for the various doses of y rays (Treatment 2). Figure 2 shows an electronic image of fluorescence from an ethidium bromide-stained gel containing supercoiled PET-2 DNA as a test DNA and BglI-restricted pUC 18 as a normalizing DNA. The quantity of supercoiled
where Fsp and Fs,o are as defined previously and FNp and FN,O are the integrated fluorescence intensities of the bands of normalizing pUC 18 DNA that received doses D and 0, respectively. The use of the normalizing DNA allows correction for lane-to-lane variation in sample application. Both gels (0 and +) gave similar results; a statistical analysis of the data is shown in Table 1. Although addition of the normalizing DNA before division of the supercoiled stock gave better results than analysis of the test DNA alone, in some experimental protocols, such an additional DNA might interfere with the reaction being studied. We thus tested whether the standardizing DNA could be added to each sample after the conversion reaction (Treatment 3). This would avoid the presence of a normalizing DNA in the supercoil cleavage reaction, but offer the advantage of its presence during quantitation. It should facilitate gel loading, but would be influenced by differences in pipetting the normalizing DNA into each sample, small volume changes during handling and irradiation, etc. Figure 1C shows that this method gave good agreement between the two independent gels, but was less accurate at low levels of conversion of supercoils (i.e., low doses). The three methods can be compared using the data shown in Table 1. First, the slopes of the regression lines are similar within the 95% confidence limit (-2~). However, Treatment 2 has the highest correlation coefficient, lowest standard deviation, and residual variance. In cases in which the addition of a standardizing plasmid during supercoil cleavage is undesirable, Treatment 3 gives reasonable standard deviation and residual variance, but does show significant scatter at low levels of supercoil conversion (See Fig. 1C). Treatment 1, in which no standardizing plasmid is used, gives a higher
ELECTROPHORETIC
QUANTITATION
OF
SUPERCOILED
DNA
CLEAVAGE
0.1 0
2
4
6
Dose
(Gy)
8
10
0
4
6
Dose
(Gy)
2
4
6
DOSE
(Gy)
8
10
10
FIG. 1. Quantitation of the conversion of supercoiled DNA to its relaxed form by three methods. (A) SupercoiledpET-2 DNA was irradiated in solution; equal aliquots of each sample were applied quantitatively to an agarose gel (Treatment 1). After electrophoresis, staining with ethidium, and destaining, an electronic image was obtained. The quantity of supercoiled DNA was determined for each sample from the integrated lane profile. The symbols (A and A) show results obtained in two independent gels. (B) Supercoiled PET-2 DNA was mixed with linear pUC 18 DNA as an internal standard or “normalizing” molecule and the solution was then divided into samples for each irradiation treatment; samples were applied to agarose gels, electrophoresed, and stained and a quantitative image was obtained (Treatment 2). The fluorescence from supercoiled PET-2 DNA and from pUC 18 DNA was determined for each sample from the integrated lane profile and the ratio of supercoiled PET-2 DNA to pUC 18 determined. The fraction of DNA that remained supercoiled at each dose was then obtained by dividing the ratio of supercoiled DNA to normalizing DNA at each dose by the value of that ratio in the unirradiated sample, as indicated in Eq. [3]. Results for two gels are shown (0, +). (C) Supercoiled PET-2 DNA was irradiated, and, after irradiation, linear pUC-18 was added to each sample separately (Treatment 3). The samples were electrophoresed, the DNA was stained with ethidium, and a quantitative image was obtained. The fraction of supercoiled DNA was calculated as in (B). The results of two independent gels are shown (0, 0).
standard deviation and residual variance, but gives a rate of cleavage that is similar to that obtained by the other methods. Quantitative loading of submerged agarose gels, however, is tedious and time consuming.
We used a variation of Treatment 2, which is described below, to quantitate the nicking of supercoiled DNA in a more complex system: cleavage of supercoiled DNA containing a T7 promoter site by the T7 RNA
84
SUTHERLAND
ET
TABLE Statistical Conditions DNA
Treatment
(standardizing addition) None Before After
1 2 3 a Standard
Comparison
deviation
of the slope
of Three ri (ssb/molecule/Gy) -0.081 -0.085 -0.079
of the dose-response
AL.
1
Methods
of Supercoil
Quantitation
$1 (ssb/molecule/Gy) 0.0261 0.0068 0.0141
Residual variance 0.0674 0.0046 0.0196
Correlation coefficient 0.975 0.997 0.992
function.
polymerase-based synthetic photoendonuclease. We chose pUC 18 as a normalizing DNA, as it does not contain a specific binding site for the polymerase. The polymerase does not bind to pUC 18 at the lower ratios of polymerase to DNA used in these experiments. T7 RNA polymerase was derivatized with an average of 17 rose bengal molecules per protein and incubated in the presence of supercoiled PET-2 DNA, which contains a strong promoter site for the polymerase, and EcoRIdigested pUC 18 as normalizing DNA. After preincubation, the samples were divided; one part of each sample was exposed to visible light (activating the rose bengal and initiating DNA cleavage), while the rest of each sample was kept in the dark. Samples were mixed with a neutral gel loading mixture containing SDS and then electrophoresed on neutral gels. After ethidium staining and destaining, a digital image was obtained using a CCD camera, and the areas of the relaxed (see below) and normalizing DNAs in each lane were determined. The fraction of supercoiled DNA converted to relaxed DNA in the light vs the fraction converted in the dark (due to non-light-dependent nicking) was calculated for each sample. Figure 3 shows that the synthetic endonuclease cleaves the DNA in samples exposed to visible light, with little DNA nicking in the absence of light exposure. Two factors complicate the analysis of supercoil nicking: first, the protein or light might nick the DNA nonspecifically, independently of their concerted endonucleolytic activity. Thus appropriate dark and light controls are required at each protein concentration. Second, measurement of initial cleavage rates requires quantitation at low levels of conversion and would thus be facilitated by measurement of the increase in relaxed molecules (a large change in an initially small number, rather than a small change in the large area of the supercoil band). We can calculate the fraction of the supercoiled molecules remaining and hence the average number of single-strand breaks per molecule induced by a certain treatment from the fluorescence intensities of only the relaxed molecules, provided that we also know the normalized intensity of the fluorescence of all of the experimental DNA-the PET-2 in our experimentswhen none of it is supercoiled. We denote this normal-
ized fluorescence intensity as FE,T/FN,T, where the “T” denotes “total” DNA in the sample and, as above, the “E” denotes “experimental” and the “N” denotes “normalizing.” The value of FE,T/FN,T can be determined in several different ways. If the value of R is known for the given experimental conditions, the value of FE,TIFN,T can be obtained from the equation F E.T
R-F,, F NT =L+F’F NJ
FRD ND
where, for the low doses that are of interest, we assume that there will be no significant number of linear molecules formed.4 Another possibility is that the ratio of the total experimental DNA to the normalizing DNA is known from the design of the experiment. A third possibility is to measure FE,-JFN,T by including in the gel a sample of the DNA mixture that has been treated with a restriction endonuclease that linearizes all the supercoiled and relaxed DNA. Combining Eqs. [3] and [4] results in the desired expression
PI If the number of breaks per molecule is linearly proportional to the extent of the treatment, a plot of the logarithm of the expression on the right-hand side of Eq. [5] will result in a straight line, the negative slope of which is the number of strand breaks per molecule per unit “dose,” where dose is used in the more general sense of “treatment.” Figure 3 shows the resulting data for cleavage by the synthetic endonuclease derivatized with 17 rose bengals per polymerase molecule. In samples exposed to 30 min visible light, the DNA was cleaved by the polymerase, with the extent of nicking dependent on the quantity of labeled polymerase added. In the absence of light, the ’ Generalizing Eq. [4] to allow for the production of linear molecules is straitforward provided that the binding and quantum yield of fluorescence of ethidium bromide to nicked circular and linear DNAs are identical.
ELECTROPHORETIC
QUANTITATION
FIG. 2. Electronic image of an electrophoretic gel showing cleavage of supercoiled PET-2 DNA by ionizing radiation. The direction of electrophoresis is from top to bottom; the bands, in order from top to bottom, are relaxed circles, linear molecules (if present), supercoiled molecules, and two BglI fragments of pUC 18, the larger of which was used as an internal standard. The supercoiled PET-2 DNA and the pUC 18 internal standard DNA were mixed, and samples were exposed to 0,0.5,1,2,4,6,8,8, and 10 Gy of 137Cs y rays, shown from left to right. The depletion of supercoiled DNA and increase in relaxed molecules contrast with the approximately constant quantities of standardizing DNA.
polymerase does not cleave the DNA. DNA without a promoter site is not cleaved significantly in the light compared to that cleaved in the dark control. It is important to consider the criteria for the selection of a normalizing DNA. First, the normalizing DNA must be easily separable from the test system in the gel system used for analysis of the test DNA. Second, the normalizing DNA must be easy to quantitate. Third, it must not interfere with the supercoil conversion reaction, nor be altered by the reaction so as to interfere with cleavage, separation, or subsequent quantitation. In the in vitro irradiations, normalizing DNA could be added to the test supercoiled DNA without consideration of its sequence. Linear pUC 18 is easily separated from supercoiled PET-2, and because of both its small size and its topology, it is less susceptible per molecule to observable effects of strand breakage. For reactions mediated by sequence-specific DNA binding proteins (such as the polymerase-based synthetic endonuclease), the normalizing DNA must not contain binding sites for the enzyme observable under the experimental reaction or gel conditions. The use of the normalizing plasmid allows facile quantitation of DNA cleavage, even at low levels of conversion. The use of a normalizing DNA can be applied to many other reactions. For example, we have used it in quantitating pyrimidine dimer formation, measured by specific nicking at dimer sites by the dimer-specific uv endonuclease from A4icrococcus luteus (12). In this case, the substrate DNA was uv irradiated, while the normalizing DNA was unirradiated. We have also used this approach to measure repair of dimers by the 40-kDa photoreactivating enzyme from Escherichia coli (13) in a
OF
SUPERCOILED
DNA
85
CLEAVAGE
coupled reaction, using uv-irradiated substrate DNA and unirradiated normalizing DNA, followed by treatment with the M. luteus uv endonuclease. The normalizing DNA method also greatly facilitates the quantitation of DNA retardation assays for measuring the binding of proteins to specific DNA sites (14). In this case, the substrate DNA contains the binding site for the protein of interest, while the normalizing DNA lacks the site. After incubation, the samples are loaded onto a neutral gel compatible with retention of DNAprotein binding and electrophoresed, and the gel is stained and destained as usual. The areas of the free (unbound) substrate DNA and the normalizing DNA are determined for each lane, and the ratio of free DNA/ normalizing DNA is determined. This method allows rapid loading of the gel (which is difficult for methods requiring quantitative transfer of gel samples) and thus maximizes the detection of labile DNA-protein complexes. We have used electronic imaging for the quantitation of fluorescence of DNA-bound ethidium because of its accuracy of DNA measurement (ll), short exposure times (minimizingethidiumphotobleaching), andconvenience. However, similar information can be obtained using photographic film, with attention to standard methods of compensating for film nonlinearity [e.g., Refs. (2-4, 15)] and failure of time-intensity reciprocity [e.g., Ref. (IS)]. Although the addition of the normalizing DNA before
E 2
4 : ;=:
1
_---
z----*
_____
---
----a i
Iv
0
4
6
12
I 16
RB-RNAP/DNA
FIG. 3. T7 RNA polymerase labeled with 17 rose bengal molecules/ protein was added to a mixture of supercoiled PET-2 DNA, which contains a T7 promoter, and BglI-linearized pUC 18, which has no polymerase binding site. The samples were preincubated with argon bubbling and then part of each sample was exposed for 30 min to visible light (O), while a companion part of each sample was kept in the dark (A). Samples were electrophoresed on agarose gels and stained with ethidium, and an electronic image was obtained, the fluorescent intensities of the relaxed and normalizing DNA bands were determined. The fraction of supercoiled DNA remaining was calculated from these intensities using Eq. [5].
86
SUTHERLAND
the partitioning of the stock solution is clearly the method of choice when possible, some reactions may not be compatible with the addition of normalizing DNAs. The data shown in Fig. 1 as well as the results of additional experiments (data not shown) allow the evaluation of the other methods that we tested. First, all three methods gave similar slopes. The residual variances in Treatments 3 (normalizing plasmid added after the conversion reaction) and 2 (normalizing plasmid added before the division of DNA stock) were similar, but the variance in Treatment 1 (no normalizing DNA) was significantly higher than that in the other two methods. Thus the method of second choice is Treatment 3, in which the reaction converting supercoils to other forms is carried out first, and then a normalizing DNA is added before gel loading. The higher variance and technical difficulty of Treatment 1 (no normalizing DNA) make it the third choice. It is clear that the use of the normalizing plasmid is the preferred method and can be applied to many reactions.
We thank Dr. Giovanni Ciarrocchi for useful Thompson for statistical analyses, and Anne Katz desi for assistance.
discussions, and Matthew
Keith Ran-
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