Photochemistry and Photobiology Vol. 44, No. 3, pp. 391 - 396, 1986 Printed in Great Britain

003 1-8655186 $03 .00+0.00 Pergamon Journal5 Ltd

QUANTITATION OF DNA DAMAGE IN NON-RADIOACTIVE DNA

BETSY M. SUTHERLAND~’*, GIOVANNI CIARROCCHI~, MARINA CIOMEI* and JOHN C. SUTHERLAND’

‘Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA and ’Istituto di Genetica, Biochimica, ed Evoluzionistica, Pavia, Italy 27100

(Received and accepted 4 February 1986)

Abstract-Three principal methods have been developed for measuring femtomoles of damage in nanogram quantities of non-radioactive DNA. Lesions which can be quantified include single and double strand breaks, alkali labile sites including apurinic and apyrimidinic sites, and pyrimidine dimers. The first in vitro method measures the conversion of supercoiled DNA to relaxed or linear molecules, and can detect up to four lesions per molecule. The second in vitro method (supercoil depletion) assesses the fraction of intact linear molecules of homogeneous length, and allows detection of 8 lesions/molecule. The third method, measurement of molecular length distributions of DNAs of heterogeneous length, reveals the extent of DNA damage and repair in vivo or in vitro.

INTRODUCTION

Measurement of damages in DNA has provided essential information on the short and long term effects of chemical and physical agents on biological systems. This approach has been limited by the requirement of radioactive labeling of DNAs, or of large quantities of nonradioactive DNA for analyses (Friedberg and Hanawalt, 1983). Recently methods for DNA damage analysis requiring nanogram quan- tities of non-radioactive DNA have been developed (Ciomei el al., 1984; Freeman et a l . , 1986). These techniques are suitable for measurement of DNA damage in vitro or in vivo, and are sensitive to as little as femtomoles of damage. Damages which can be quantified directly include the single strand break, double strand breaks, and alkali labile sites (apurinic or apyrimidinic sites). Moreover, the existence of lesion-specific endonucleases, such as the UV en- donuclease specific for pyrimidine dimers (Ahmed and Setlow, 1979) allows quantification of the lesions through measurement of the resulting single strand breaks (Achey et al., 1979).

Three major methods for DNA damage detection include (1) measurements of the conversion of super- coiled covalently closed circular DNA to a relaxed molecule (a double-stranded circle containing at least one nick in one strand), (2) the supercoiled depletion method, and (3) determination of the distribution of a heterogeneous DNA population and calculation of the extent of the population dispersion, by the number average, weight average or median molecu- lar length. We shall discuss the applications and limitations of these methods, and their advantages and drawbacks in DNA damage determinations.

MATERIALS AND METHODS

DNA. T7 bacteriophage DNA was prepared by phenol

*To whom correspondence should be addressed. ~~

extraction of purified virus, followed by dialysis against 10 mM Tris, pH 8, 50 mM NaCI, 0.1 mM EDTA.

Plasmid pBR322 was purchased from Bethesda Research Laboratories or prepared by lysozynie treatment of chlor- amphenicol-amplified Escherichia coli HMS 174 containing the plasmid, and plasmid DNA isolation on a Cs Cl- ethidium bromide gradient, followed by dialysis as above.

Radiation sources and dosimetry. Ultraviolet radiation (180-300 nm) was monitored with a pyroelectric detector (Molectron, Inc.). In some experiments, a Johns monochromator containing a high pressure Hg lamp [(mon- itored with a silicone photodiode (EG and G , Inc.)] or a 15 W germicidal lamp (G15T8) monitored with a Jagger meter (Jagger, 1961) were used as radiation sources. Gamma radiation from a I3’Cs source was monitored with a Victoreen ion gauge.

Gels. Agarose (Sigma, Type 11) gels, 0.4% or 1% (wt/vol) in 1 mM EDTA, 50 mM NaCI, were poured in BioRad 6.4 cm X 10 cm Mini Sub-cell trays with 15 well combs (2 X 0.75 mm wells). The alkaline gel electrophoresis buffer was 2 mM EDTA, 30 mM NaOH (McDonell et al., 1977); electrophoresis was carried out for 2 h at 30 V, in a neutral buffer of 89 mM Tris-borate buffer, pH 8.5 and 8 mM EDTA. After electrophoresis, the alkaline gels were neutralized by immersion in 0.1 M Tris, at pH 8 for a minimum of 30 min. The gels were stained in 0.5 & n e ethidium bromide in water for 15 min, rinsed and excess stain removed by leaving them for at least 15 min in water. Gels were placed on a UV Products Transilluminator, and photographed through a red filter, using Polaroid N/P 55 film.

RESULTS AND DISCUSSION

We used three principal systems for analysis of lesions in DNA by agarose gel electrophoresis. Each system has its own special advantages and limita- tions. The first system depends on the conversion of supercoiled covalently closed circles of double- stranded DNA to linear molecules by the introduc- tion of double strand breaks, or to relaxed circles (DNA duplexes containing one linear strand and one closed circle) by the introduction of one or more single strand breaks. Figure 1 shows the three DNA conformations; since they differ in shape (but not in molecular length), the supercoiled, relaxed and

391

392 BETSY M. SUTHERLAND et al.

linear molecules have different mobilities during electrophoresis in agarose and can be separated easily (McDonell et al., 1977). Panel A of Fig. 2 shows a separation of three conformations of the plasmid pBR322: lane a contains principally super- coiled molecules (the rapidly migrating, heavier band) and a more slowly migrating minor component of relaxed circles. Lane b shows the migration of linear DNA molecules, in this case produced by the action of Bam HI, a restriction endonuclease with a single cleavage site per pBR322 monomer. Lane c shows the migration of relaxed circular DNA, pro- duced from supercoiled DNA by the action of an endonuclease that cleaves single strands.

Since one double strand break converts a super- coiled molecule to the linear form, the number of double strand breaks introduced into a population of molecules can be determined from the proportion of molecules containing a known number of nicks. Figure 2 (Panel B, lanes a-h) shows DNA popula- tions with increasing fractions of molecules with double strand breaks. The supercoiled DNA is a dimeric-length pBR322; the first double strand cleav- age by a restriction enzyme with one cleavage site per pBR322 monomer converts the supercoiled dimer to a linear dimeric molecule. The second cleavage converts linear dimers to linear monomers. The quantification of the number of molecules in the supercoiled and linear conformations requires a proportional response of the densitometer to DNA quantity. Sutherland et al. (1984) showed that within a range of DNA concentration, the area under the peaks calculated by their computer-controlled scan- ner is indeed a linear function of the amount of DNA on the gel.

Double strand breaks are introduced into DNA by a variety of agents: direct and indirect action of ionizing radiation, high doses of ultraviolet radiation, and radical-mediated reactions, as well as by en- zymes such as the restriction enzyme Bam HI. If the number of scissions per molecule is small, this method provides a quick, accurate method of analys- ing double strand breaks in nanogram quantities of non-radioactive DNA.

For the quantitation of single strand breaks, the experimental approach is similar to that for double strand breaks, but the analysis is more complex. Since both one single strand break and a few breaks convert supercoiled molecules to the relaxed form (so that relaxed molecules containing one break cannot be distinguished from those containing a few breaks), the fraction of the two types of molecules must be obtained from the raw densitometric data by use of Poisson statistics (Kuhnlein et al., 1976). In addition, the number of nicks in the initial population must be taken into account. Figure 3 shows the introduction of 0.26 to 2.06 fmol of single strand breaks into the supercoiled DNA.

Single strand breaks are produced in DNA by ionizing radiation, high doses of ultraviolet radiation and the action of specific and non-specific nucleases.

Figure 1. Three conformations of double stranded DNA: supercoiled (covalently closed circle containing at least one superhelical twist), relaxed (one single strand circular molecule and its complement, containing at least one nick in one of the two strands), and linear (double strand, non-

circular molecule).

a b c

a b c d e f g h

Figure 2. Agarose gel containing pBR322. Panel A: lane a, principally supercoiled DNA with a small component of relaxed molecules; b, linear DNA; c, relaxed molecules. Panel B: Time course of digestion of pBR322 dimer by the restriction enzyme Barn HI (a, 0’; b, 1’; c, 2‘; d, 5’; e, 10‘; f ,

20’; g, 60’) Arrows indicate direction of migration.

The existence of nucleases which introduce single strand breaks at the site of specific DNA damages allows quantitation of such damages. A nuclease specific for cis-syn pyrimidine dimers, the Micrococ- cus luteus UV-endonuclease (Ahmed and Setlow, 1979) allows us to determine dimer levels in super- helical DNA. The data in Fig. 3 reflect the formation of dimers in pAT153 DNA by 254 nm radiation.

In addition to frank double or single strand breaks, radiation or chemical treatments also can produce alkali labile sites. Alkali labile sites are DNA lesions which do not result in phosphodiester bond breakage under neutral conditions but yield strand breaks after treatment at high pH. An example is base loss, resulting in an apurinic or apyrimidinic site. The hallmark of this lesion in supercoiled DNA is the appearance of relaxed molecules upon elec-

DNA damage measurement 393

Figure 3. Neutral agarose gel containing pAT153. The left most lane contains UV irradiated DNA not treated with UV endonuclease. All other lanes contain DNA treated with the endonuclease; pyrimidine content of these samples (from left to right) is 2.06,1.85,1.52,1,0.51,0.25,0.2,O.23, 0.2 and 0.26 fmol. The arrow indicates direction of migra-

tion.

trophoresis in alkaline gels, but not in neutral gels. Like all techniques that quantify DNA breakage,

the supercoiledirelaxedllinear measurement method demands that there is minimum introduction of extraneous breaks into the DNA during handling and storage. These conditions are best met by the use of sterile buffers, tubes and pipettes, gentle handling and storage of DNA in sterile buffer at 4°C (freezing and thawing can introduce single strand nicks). In a 2 x 0.75 mm well in a Bio-Rad Mini-Sub Cell gel electrophoresis apparatus, 5 ng of plasmid DNA easily can be visualized; this amount corresponds to a total of about 20 fmol of DNA molecules. Conver- sion of 25% of the molecules from supercoiled to relaxed molecules can be detected, therefore about 5 fmol of DNA damage can be quantitated readily. This technique has the advantage of using commer- cially available non-radioactive DNA. The proce- dure requires determination of quantities of DNA in discrete bands; as long as one works in a DNA concentration range which produces proportional darkening of photographic film, this can be obtained easily as the area under the peak by commercial scanning densitometers. Since the first break in a molecule can be easily detected, the sensitivity of the method can be increased by simply using a larger supercoiled molecule; for example, multimers of pBR322 allow the detection of one half (for the dimeric length molecule) or one third (for the trimer) as many lesions as when we use the monomer.

The limitations of the method include the sensitiv- ity of supercoiled DNA to minute levels of nucleases and nicks caused by handling; in this respect, the larger DNAs are especially labile. Double strand break analysis allows the quantitation of the first break per molecule; at higher numbers of breaks, a smear of small DNA fragments appears which can not be analysed by this method. Since this procedure requires supercoiled DNA for the initial DNA population, it precludes the study of lesions in single stranded DNA. For single strand break detection we can measure up to four sites per molcule by this method.

To increase the number of sites per molecule which can be measured in supercoiled DNA, Ciomei et al. (1984) developed a supercoiled DNA depletion method. Figure 4 illustrates the principles of this method: Supercoiled DNA is treated with radiation, chemical or other agents, the DNA then is cleaved PAP 4413 K

with a restriction enzyme having only one restriction site in the DNA, to yield a population of linear molecules of homogeneous length. The DNA also may be cleaved at the same time with lesion-specific enzymes. The resulting DNA is subjected to elec- trophoresis in alkali; the intact DNA appears as a principal band (denatured supercoil) with a smear of smaller linear fragments preceding the supercoil. The photographic negative is scanned, and the amount of intact DNA is determined. Figure 5 shows the results of such an experiment.

This method detects as many as eight sites per pAT153 supercoiled molecule. Since the analysis depends on the determination of the amount of DNA i n a discrete , homogeneous band, a n a rea- determining program of a commercial densitometer can be used for quantitation (again with the require- ment for a known relationship between DNA con- centration and film darkening). The limitations of the method are the requirement for double stranded supercoiled molecules as a source of a population of homogeneous DNAs, precluding the examination of lesions in single stranded DNA or cellular DNA. In addition, the number of DNA alterations must be small enough not to interfere with supercoil lineariza- tion by restriction.

To complement these two methods for the apprais- al of lesions in supercoiled DNA, we developed a general technique for analysing lesions in non- radioactive DNA which does not require DNA populations of supercoiled or entirely homogeneous- length linear molecules (Sutherland et al., 1980; Freeman et al . , In press). The principle of the methodology is presented in Fig. 6. The upper panel shows the production of double strand breaks in linear DNA molecules; Fig. 7 , Panel A, illustrates the appearance of DNA containing double strand breaks after electrophoresis under neutral condi- tions.

Figure 6, Panel B shows the principle of single strand break analysis in linear DNA. On a neutral gel DNA molecules containing single strand breaks are comparable in molecular weight to intact molecules. However, upon electrophoresis in alkali, in which the complementary DNA strands separate, the single strand breaks result in the appearance of smaller fragments, seen as “smear” of DNA with higher mobility than the intact DNA (Fig. 7 , Panel B).

Quantitation of the number of strand breaks includes four main steps: (1) visualization of the DNA, (2) quantitation of the DNA at each point along the gel, (3) determination of the average molecular length of a treated population, and (4) comparison with the average molecular length of an untreated DNA population. DNA is visualized by neutralizing the alkaline gels to allow the DNA to renature, and then soaking the neutralized gel in ethidium bromide. Ethidium intercalates into double stranded DNA and, in such a non-polar environ- ment, its fluorescence quantum yield increases great- ly. Thus double stranded DNA in the gel can be easily

394

treoted

BETSY M. SUTHERLAND et al.

I

lesion

t rea tment with r . e and x-specif ic J-> re o g e n t

T R E A T E D DNA L I N E A R , CLE AVED

untreatel ' Figure 4. Experimental design of supercoiled DNA depletion method. Supercoiled DNA containing one site for a restriction enzyme, and the same DNA treated to contain lesion X, are digested with the pertinent restriction enzyme and a reagent producing a nick adjacent to lesion X. Upon alkaline gel electrophoresis the single strands separate; untreated DNA remains as a homogeneous population of linear molecules, while treated DNA shows a distribution of smaller molecules resulting from cleavage

at lesion X.

a b c d e f g h

Figure 5. DNA damage detection by the supercoiled DNA depletion method. Electrophoretic pattern of alkaline agar- ose gel of pAT153 RFI DNA linearized with Eco RI and digested with M . luteus pyrimidine dimer endonuclease DNAs in lanes c, d, e and f containing 0 ,4 ,8 and 16 dimers per mol, respectively. Lanes a, h and b, g contain pAT153 restricted with EcoRI and BamHI (a, h) or with Bgl I (b, g) as position and intensity markers. The arrow indicates

direction of migration.

ANALYSIS OF DOUBLE S T R A N D BREAKS DNA

intact w i t h DNA drb

Q b

ANALYSIS OF SINGLE S T R A N D BREAKS

DNA l n t o t i w i t h DNA ssb neutrol

0 b

Figure 6. Principles of analysis of double and single-strand breaks in DNA populations of heterogeneous lengths. Upper panel: DNA conformation and expected gel elec- trophoretic pattern for intact DNA (a) and DNA containing double strand breaks (b). Lower panel: conformations and expected neutral and alkaline gel electrophoretic pattern for intact DNA (a) and DNA containing single strand

breaks (b).

detected by the fluorescence of bound ethidium. DNA is quantitated by photographing the fluoresc- ence, and scanning the resulting negative with a computer-controlled scanner. Since the film response is non-linear, the amount of film darkening is convo- luted with the film-response function to yield DNA quantity corresponding to a given mobility. The resulting distribution of DNA quantity as a function of electrophoretic mobility is compared with the mobility of molecular length markers to obtain the

DNA damage measurement 395

A

B

a b c d e f g h

a b c d e f g h i j k l m n

Figure 7. A. Electrophoretic pattern of neutral gel containing 'I7 DNA either unirradiated (lanes b, d, g) or exposed to 800 rad or y radiation (lane c, e, h). Lane a shows molecular length markers of intact T7 DNA plus the three Bgl I cleavage products of T7 DNA (40, 23, 14 and 4 kb, respectively). The arrow indicates direction of migration. B. Electrophoretic pattern of alkaline agarose gel containing T7 DNA treated (lanes d, f , h, j, 1) or untreated (lanes c, e, g, i, k) with the pyrimidine dimer-specific UV endonuclease after exposure to 0 (lanes c and d), 5 (e and f), 10 (g and h), 20 (i and j ) or 30 (k and 1) Jimz of 254 nm radiation. Lanes a and m contain T7 DNA plus T7 restricted with Bgl I; lanes

b and n contain T7 restricted with PvuII. The arrow indicates direction of migration.

xiet-1

P DISTfiNCE ~ 7 . 9 1 sMOLECULfiR LENGTH +49

F L U 0 R E s C E N C E

-5 e f ie 15 28 2s 38

DISTINCE inti>

Figure 8. Densitometric scan of molecular length markers from lowest to highest mobility: T7, (40 kb); the three Bgl I cleavage fragments of T7 (23, 14 and 4 kb respectively).

396 BETSY M. SUTHERLAND et al.

number average molecular length of the DNA population (Fig. 8). The number of breaks induced by a given treatment of a population of DNA molecules then is calculated from the equation

1 1 N = - - - LN, treated LN, untreated

where N is the number of sites per kilobase, and LN, treated and L N , untreated are the number average molecular lengths of the treated and untreated populations, respectively.

Advantages of this method include the ability to study low levels of DNA damage in nanogram quantities of non-radioactive, non-supercoiled high molecular weight DNA populations of heter- ogeneous length. Double strand breaks can be mea- sured by electrophoresis under non-denaturing con- ditions, while alkali labile sites and single strand breaks can be assessed by electrophoresis in alkaline gels. Breaks induced directly by chemical or physical agents, by restriction enzyme cleavage or by lesion- specific endonucleases also can be measured.

The sensitivity of the method depends on a good separation of intact molecules from smaller frag- ments. Two principal factors influencing the separa- tion are the composition of the gel, with higher molecular weight fragments requiring lower percen- tage agarose, and the conditions of electrophoresis. High voltage gradients induce reptilian motion of large DNAs, producing loss of resolution of high molecular weight species (McDonell et al., 1977). In addition to selection of appropriate voltage gra- dients, cooling and recirculation of the elec- trophoresis buffer also improve resolution.

In our current system, 3&50 ng of high molecular weight DNA can be easily quantitated. The quantity depends on the size of the gel and sample loading well; we use gels that are 6.4 x 10 cm, and the wells are 2 x 0.75 mm. Further reduction in DNA quantity can be achieved by using a smaller well, although some difficulty may be encountered in loading the sample. It is essential that the correct quantity of DNA for each DNA distribution be placed on the gel; we developed a simple, non-destructive method of determining DNA concentrations in sub- microliter samples (Moore and Sutherland, 1985). Since the quantity required also depends on the degree of heterogeneity of the sample, it is often useful to run a preliminary gel with part of each sample.

These three methods, then, allow the detection of DNA damage in small quantities of non-radioactive DNA. The first, measurement of supercoiled, re- laxed and linear molecules, requires little DNA and the analysis can be carried out using a commercial densitometer. It is limited to measurement of about four lesions per molecule. The second, the supercoil

depletion method, has the same advantages as the first method, but detects as many as eight lesions per molecule. Its major difficulty is in distinguishing intact molecules from very large cleavage fragments. The third method, the determination of number average molecular weight of heterogeneous DNA populations, permits the study of DNA damage and repair in nanogram quantities of cellular DNA in vivo or purified DNA in vitro. Analysis can be carried out by densitometric scanning and gravimetric analysis, but is obtained more conveniently by computer analysis. Future developments in the methodology may include increased speed of analysis, higher sensitivity of lesion detection and extension of the range of lesions detectable.

Acknowledgements-We thank Ms. A. Shih and Ms. A. Katz for excellent technical assistance. This research was supported by the US Department of Energy, grants from the National Cancer Institute (CA26492 and CA23096) to BMS, and by the Progetto Finalizzato del Consiglio Nazionale delle Ricerche "Oncologia".

REFERENCES A c h e y , P . M . , A . D . W o o d h e a d and R . B .

Setlow (1979) Photoreactivation of pyrimidine dim- ers in DNA from thyroid cells of the teleost, Poecilia formosa. Photochem. Photobiol. 29, 307-310.

Ahmed, F. E. and R . B . Setlow (1979) DNA Repair in xeroderma pigmentosum cells treated with combinations of ultraviolet radiation and n-acetoxy-2-acetylamino- fluorine. Cancer Rex 39, 471-479.

C i o m e i , M . , B . M . S u t h e r l a n d a n d G . Ciarrocchi (1984) Quantification of pyrimidine dim- ers and apurinic sites in DNAs of uniform length. Photochem. Photobiol. 39, 593-596.

Freeman, S. E. , R. W. Gange, E . A. Matzinger and B. M. Sutherland (1986) Higher pyrimidine dimer yields in skin of normal humans with higher UVB sensitivity. J . Inv. Derm. 86,-34-36.

Friedberg, E. C. and P. C. Hanawalt (1983) DNA repair: A laboratory manual of research procedures, Vol. 2. Dekker, New York.

Jagger, J. (1961) A small and inexpensive ultraviolet dose-rate meter useful in biological experiments. Radiat. Res. 14, 394-403.

Kuhnlein, U., S. Penhoet and S. Linn (1976) An altered apurinic DNA endonuclease activity in group A and group D xeroderma pigmentosum fibroblasts. Proc. Natl. Acad. Sci. USA 73, 1169-1173.

McDonell, M. , M . N. Simon and F. W . Studier (1977) Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis of neutral and alkaline gels. J . Molec. Biol. 110, 119-143.

Moore, S. P. and B. M. Sutherland (1985) A densi- tometric nondestructive micro assay for DNA quantita- tion. Anal. Biochem. 144, 15-19.

Sutherland, B . M., I . Kochevar and L. Harber (1980) Pyrimidine dimer formation and repair in human skin. Cancer Res. 40, 3181-3185.

Sutherland, J. C., D . C. Monteleone, J. C. Trunk and G . Ciarrocchi (1984) Two-dimensional, computer- controlled film scanner: quantitation of fluorescence from ethidium bromide-stained DNA gels. Anal. Biochem. 139, 390-399.