Double Strand Breaks Induced by Low Doses of γ Rays or Heavy Ions: Quantitation in Nonradioactive Human DNA

ANALYTICAL BIOCHEMISTRY 239, 53–60 (1996)ARTICLE NO. 0290

Double Strand Breaks Induced by Low Doses of g Rays orHeavy Ions: Quantitation in Nonradioactive Human DNA1

Betsy M. Sutherland, Paula V. Bennett, and John C. SutherlandBiology Department, Brookhaven National Laboratory, Upton, New York 11973

Received January 24, 1996

may sediment anomalously in neutral gradients (12,We have developed a method of quantitating low fre- 24, 25); furthermore, the rate at which large molecules

quencies (0–30 sites/109 base pairs) of double strand elute from filters at neutral pH can be affected by ex-breaks in Ç1 mg of nonradioactive human DNA. Unir- trinsic factors (5, 6, 13, 18, 22, 33, 50).radiated or irradiated DNA is digested with the re- Contopoulou et al. first used pulsed-field gel electro-striction endonuclease NotI, producing cleavage frag- phoresis to observe generation of double strand breaksments that include a major group centered atÇ1.2–1.3 in Saccharomyces cerevisiae cells by ionizing radiationMbp. The DNA molecules are separated as a function (11). Taking advantage of the fact that very large mam-of size by transverse alternating field electrophoresis. malian DNAs do not enter standard neutral electropho-The frequency of double strand breaks is computed retic gels even under pulsed-field conditions, empiricaldirectly from the decrease in number average molecu- methods have been developed for measuring strandlar length induced in the 1.2- to 1.3-Mbp cleavage frag-

breaks (2, 4, 19, 23, 28, 35–37, 39–41). Radiation-in-ment group by 137Cs g or Fe26/ (1.1 GeV/nucleon) irradi-duced strand breaks reduce the molecular length suffi-ation vs the corresponding unirradiated DNA samples.ciently such that a portion of the DNA enters the gel.The double strand break frequency can be quantitatedThe increase in the fraction of DNA entering the geleasily in the dose range of 0–10 cGy of g rays. Theis then used as a measure of strand break formation.frequency of breaks per unit dose calculated for g irra-However, the fraction of DNA remaining in the well,diation of DNA in human cells (Ç4.6 double strandas estimated from the fluorescence of bound ethidium,breaks/109 bp/Gy) is within the range of values ob-does not agree with that determined from radiolabelingtained by others (2–8 sites/109 bp/Gy) who used meth-(52). Since differences in the environment of DNA inods requiring higher doses. q 1996 Academic Press, Inc.

the well compared to that in the gel are more likely toaffect fluorescent labels than radiolabels, many investi-gators limit this method to radiolabeled DNA.

Substantial evidence indicates that unrepaired dou- Several investigators cleaved DNA with rare-cuttingble strand breaks (DSBs)2 are major lethal and muta- restriction enzymes, electrophoresed the resultinggenic lesions induced by ionizing radiation in mamma- cleavage fragments using a variety of gel modes, usedlian cells [for a review see Ward (51)]. Although many radiolabeled probes to known fragment(s), and, assum-methods for quantitating DSB have been developed, ing a Poisson distribution of breaks, calculated the frac-most provide data only at high frequencies of DSB, tion of the intact band depleted by radiation-inducedcorresponding to large, lethal doses. Quantitating DSB double strand breaks (29, 31). These methods allowat low doses is difficult because of the behavior of very measurement of double strand breaks at moderate fre-large DNAs from mammalian cells: large molecules quencies, but generally are limited to g-ray doses of

úÇ1 Gy. Using contour-clamped homogeneous electricfield (CHEF) electrophoresis and hybridization to an1 The U.S. Government’s right to retain a nonexclusive royalty-freeappropriate probe, Nevaldine et al. obtained relativelicense in and to the copyright covering this paper, for governmental

purposes, is acknowledged. DSB yields at doses as low as 25 cGy (31). Cedervall et2 Abbreviations used: DSBs, double strand breaks; CHEF, contour- al. compared four methods of calculating double strand

clamped homogeneous electric field; Mbp, megabase pairs; Gbp, giga- break frequencies induced by radiation doses greaterbase pairs; NFFs, neonatal foreskin fibroblasts; TE, Tris–EDTA; IC,than Ç5 Gy (9).ionization chamber; TAFE, transverse alternating field electrophore-

sis; CCD, charge-coupled device. To quantitate DSBs in small amounts of nonradioac-

530003-2697/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

AID AB 9603 / 6m1a$$$181 06-19-96 06:30:34 aba AP: Anal Bio

SUTHERLAND, BENNETT, AND SUTHERLAND54

tive DNA exposed to low radiation doses, we sought to times in room temperature (237C) phosphate-bufferedsaline (0.17 M NaCl, 3.4 mM KCl, 10 mM Na2PO4, 1.8develop a method that would allow determination of

the DSB break frequency directly from the length dis- mM KH2PO4).tribution of DNA molecules in each population. Over Preparation of human DNA. All solutions, equip-the past 15 years we have developed sensitive methods ment, and materials were sterilized by appropriateusing this approach for quantitating DNA lesions af- means. All manipulations were carried out using pow-fecting one strand (14, 42, 44, 46–49). This method der-free gloves. Cells were suspended in 1 vol 20 mMuses agarose gel electrophoresis of nanogram quanti- NaCl, 0.1 M EDTA, 10 mM Tris–Cl, pH 8.3 (lysisties of nonradioactive DNA, followed by ethidium stain- buffer), and mixed with 3/4 vol 2% InCert agaroseing, and quantitative electronic imaging. The number (FMC, Rockland, ME) melted in lysis buffer and cooledaverage molecular lengths of the DNA populations are to 377C after melting, yielding 4 1 106 cells/ml in 0.85%calculated by the method of moments and from them agarose. Fifty microliters of the cell/agarose suspensionthe lesion frequencies. Direct calculation of the number was pipetted into each well (2.5 1 1.5 1 10 mm) of aaverage molecular lengths is a general approach for microplug mold (Aquabogue Machine Shop, Aqua-analyzing DNA lesion frequencies, not requiring the bogue, NY) and solidified for 1 h at 47C. Plugs wereassumption of a random or Poisson distribution of sites. transferred to a 60-mm plastic petri dish, treated for 4

In this method, the initial lengths of the molecules in days with 10 vol proteinase K (Boehringer Mannheim,the DNA population determine the sensitivity of lesion Indianapolis, IN), 1 mg/ml, 0.2% n-lauroyl sarcosine inquantitation. Thus, it seemed that NotI digestion of lysis buffer with daily changes of proteinase K solution,human DNA, which produces a group of cleavage frag- washed twice with 10 vol TE (10 mM Tris–Cl, pH 7.5,ments centered in the 1.2 to 1.3 Mbp range (megabase 1 mM EDTA), soaked twice for 20 min in 10 vol TE pluspairs, 106 base pairs) (15, 38), might provide a high- 40 mg/ml phenylmethylsulfonyl fluoride and then twicemolecular-length subpopulation ideal for sensitive in 10 vol TE for 1 h. The plugs (Ç1 mg DNA/plug) werequantitation of DSBs. The NotI cleavage fragments, transferred to a tube containing 2 ml of 150 mM NaCl,along with molecular length standards, would be elec- 10 mM Tris–Cl, pH 7.9, 10 mM MgCl2, 0.01% Tritontrophoresed in an agarose gel using an appropriate X-100 and soaked for 1 h; the soaking was repeatedelectrophoretic regime to disperse the DNAs according with fresh solution. The solution was removed, andto molecular length. A DNA dispersion curve would plugs were soaked overnight in 1 ml NotI buffer (150then be calculated from the molecular length stan- mM NaCl, 10 mM Tris–Cl, pH 7.9, 10 mM MgCl2, 1 mMdards, the number average molecular lengths calcu- DTT, 100 mg/ml bovine serum albumin). The buffer waslated for each DNA population, and, from them, the removed from the plugs and 500 ml of freshly preparedfrequency of DSBs (14). This approach allows quantita- NotI buffer containing 35 U of NotI (New England Bio-tion of double strand breaks in human DNA irradiated

Labs, Beverly, MA) was added. Plugs were incubatedwith doses of 137Cs g rays or Fe26/ ions (1.1 GeV/nu-30 min on ice and then 6 h at 377C and an additionalcleon) as small as 2 cGy, in the range of lesion frequen-35 U NotI was added and digestion was continued over-cies of 0–30 DSB/Gbp (Gbp, gigabase pair, 109 basenight. The plugs were washed twice (1 h, 3 ml TE) andpairs).then five times (1 h, 5 ml 10 mM NaPO4 buffer, pH 7.2),soaked overnight in fresh phosphate buffer, and placed

MATERIALS AND METHODS separately into tubes containing 1 ml ice-cold NaPO4

buffer.Cell culture. Exponentially growing human cells,Irradiation. Plugs were irradiated on ice with 0, 2,GM4429B (SV40 transformed xeroderma pigmentosum

5, 10, 50, or 100 cGy of g radiation from a 137Cs sourcecells) from the NIGMS Human Genetic Mutant Cellcalibrated using a Victoreen meter or lithium fluorideRepository or primary cultures of neonatal foreskin fi-thermoluminescent dosimeters. Other plugs were irra-broblasts (NFFs) established in our laboratory (CIRCdiated at room temperature with 0–50 cGy Fe26/ ions152, approved by the Brookhaven Human Studies Re-(1.1 GeV/nucleon) from the Brookhaven National Labo-view Committee) were grown in (for GM4429B) mini-ratory Alternating Gradient Synchrotron during themal essential medium (Gibco/BRL, Gaithersberg, MD)1995 radiobiology heavy ion run. The dose was calcu-plus 10% fetal bovine serum (Hyclone, Logan, UT), orlated from the output of a segmented gas-filled ioniza-for NFFs, a 1:1 (v/v) mixture of Dulbecco’s modifiedtion chamber (IC). The charge produced by heavy ionsEagle’s medium prepared in our laboratory (43) andpassing through the IC was integrated and convertedMCDB 153 (Sigma, St. Louis, MO), supplemented withto a number of counts on a scaler that was read by a50 mg/ml human recombinant insulin (Gibco), 25 mg/mldedicated dosimetry computer. The relation betweenbovine transferrin, 33 mg/ml selenium (Sigma), anddose and number of counts was given by the known10% fetal bovine serum. Cells were treated with trypsin

and centrifuged, and cell pellets were washed three energy deposition properties of heavy ions in the IC


LOW-FREQUENCY DOUBLE STRAND BREAK QUANTITATION 55

gas; when the number of scaler counts reached the duces double strand breaks, resulting in the appear-ance of smaller DNA fragments at the lower molecularnumber calculated to correspond to the desired dose,

the computer sent a beam cutoff signal to the accelera- length edge of the 1.2- to 1.3-Mbp NotI cleavage group.To determine the frequencies of DSBs by calcula-tor to terminate the exposure. The flux across the area

of the sample holder was determined by exposure of a tion of number average molecular lengths, one mustproceed as follows: first, select a DNA population forphosphor storage plate (Molecular Dynamics, Sun-

nyvale, CA) that was read by a Molecular Dynamics analysis, e.g., the 1.2- to 1.3-Mbp NotI cleavagegroup; second, determine the relationship betweenModel 425B PhosphorImager and quantitated using

Molecular Dynamics ImageQuant software. DNA size and position on the gel (the DNA dispersionfunction of the gel); and third, obtain a measure ofGel electrophoresis. After irradiation, plugs werethe mass of DNA at each position in the experimentalwashed three times, 1 h each, in 1 ml 11 TAFE bufferlanes in the population to be analyzed. The migration(10 mM Tris, 87 mM acetic acid, 0.5 mM EDTA-freeposition and known molecular lengths of S. cerevisiaeacid, pH 8). l ladders (Bethesda Research Laboratories,and H. wingei chromosomal DNAs and concatamersRockville, MD) and chromosomes of S. cerevisiae,of bacteriophage l were used to construct an electro-YPH80 (Clontech, Palo Alto, CA) [length assignmentsphoretic dispersion function for each gel, e.g., Fig.according to Gerring et al. (17)] were molecular length2. The data points were fit with a spline functionstandards; for some gels, chromosomes of Candida al-(Microcal Origin, Microcal, Northampton, MA). Mo-bicans (Clontech), Hansenula wingei (Bio-Rad, Her-lecular length standards were chosen to span acules, CA), and a mixture of l DNA, T7 DNA, and agreater range than the experimental samples to pro-BglI digest of T7 provided additional size standards.vide a more robust DNA dispersion function.Plugs were loaded into wells of a 1% TAFE agarose

The relative mass of DNA at each position also mustgel [PFGE agarose (Amresco, Solon, OH) in 11 TAFEbe determined. To provide accurate DNA quantitation,buffer]. Plugs were sealed into the wells with 1% agar-the fluorescence of ethidium bromide bound to DNAose. Electrophoresis was on a Beckman GeneLine Iwas measured using a CCD-based system designed andtransverse alternating field electrophoresis apparatusverified to provide uniform UV transillumination, mini-(TAFE, Beckman Instruments, Fullerton, CA) ener-mize vignetting, and quantitate DNA with a linear re-gized by a laboratory-built switching power supply (30sponse over at least three decades (46, 49).min, 4 s pulse, 90 V between electrodes; then 10 min,

Figure 3 shows DNA profiles for the 1.2- to 1.3-Mbp4 s pulse, 190 V; then 17–18 h, 60 s pulse, 190 V).cleavage groups in lanes 1 (unirradiated DNA, solidDNA quantitation. Gels were stained for 15 minline) and 3 (DNA irradiated with 4 cGy Fe26/ ions, dot-in ethidium bromide (1 mg/ml) in distilled water andted line) in Fig. 1. The integration limits were chosendestained as long as overnight and an electronic imageto correspond to the largest and smallest moleculeswas obtained using a second generation laboratory-within a profile that represent signal from ethidiumbuilt charge-coupled device (CCD)-based system (46,bound to DNA, that is, the positions at which the DNA49). A photographic image on Polaroid 57 film was alsosignal exceeded the background fluorescence in that gelobtained for archival purposes.region. Although the samples in Fig. 1 were arrangedin order of increasing dose for clarity for publication,

RESULTS samples on routine gels were arranged so that the oper-ator did not know their identities. The starting andFigure 1 shows a TAFE gel containing NotI-digested

human DNA irradiated with increasing doses of Fe26/ ending points of the integration for the unirradiatedDNA were pixels 143 to 210, whereas those for theions (1.1 GeV/nucleon), along with molecular length

standards. Figure 1a shows a photograph of the gel, 4-cGy sample were pixels 144 to 244. The reciprocalnumber average molecular length, (Ln

01), of each DNAwhich was used for archival purposes only and not forquantitation. For number average length quantitation, distribution is calculated from the equationa quantitative electronic image of the same gel wasobtained independent of the Polaroid photograph (Fig.1b). Three DNA size groups appear in the experimental

L01n Å

*r(x)rdx

L(x)

* r(x)rdx, [1]lanes: first, the largest molecules that remain in or

near the well; second, a group centered at 1.2–1.3 Mbp(in the size range of the S. cerevisiae 1.6- to 0.76-Mbpchromosomes); and third, smaller fragments centered where L(x) is the length of the DNA molecules that

migrated to position x, and r(x) is the intensity of ethid-at about 0.35 Mbp. Figure 1c shows an enlargement ofthe Ç1.2- to 1.3-Mbp NotI cleavage group for lanes 1– ium fluorescence from DNA molecules that have mi-

grated to position x (14). The frequency of DSBs is then5, corresponding to doses of 0, 2, 4, 8, and 16 cGy. Thefigures clearly show that increasing irradiation pro- calculated from the equation



FIG. 1. Fluorescence of ethidium bromide bound to heavy ion-irradiated NotI-digested human DNA electrophoresed along with molecularlength standards on a transverse alternating field gel. (a) Polaroid photograph. DNA from NFF cells (lanes 1–7) was exposed to 0, 2, 4, 8, 16,24, or 50 cGy of Fe26/ ions (1.1 GeV/nucleon). Lane 8 contains l ladders; lane 9, S. cerevisiae chromosomes; lane 10, H. wingei chromosomes.(The Polaroid photograph was used for archival purposes only and was not used to obtain quantitative data.) (b) Intensity color-encodedquantitative electronic image of the same gel as in a. The sizes of the S. cerevisiae chromosomes are shown at the right. After electrophoresis,the gel was stained with ethidium and destained, and a quantitative electronic image was obtained using a charge-coupled device-based camerasystem (45, 46, 49). For quantitative analysis, the electronic image was displayed in intensity-encoded color using a rainbow transform (low tohigh: light purple, dark purple, blue, green, yellow, orange, and red) to maximize visualization of the range of DNA quantities. (c) Enlargementof the 1.2- to 1.3-Mbp NotI cleavage group in lanes 1–5; the positions of the S. cerevisiae chromosomes in this size region are also shown.

ated molecules (14). Since the integrals in the numera-f Å L01n (/rad) 0 L01

n (0rad), [2]tor and denominator of Eq. [1] are, respectively, propor-tional to the first and zeroth moments of the molecularwhere f is the frequency of double strand breakslength distribution function (8), this procedure for de-(breaks per unit length of DNA) induced by the radia-termining Ln and f is called the method of moments.tion, Ln(/ rad) is the number average length of the

The upper points in Fig. 4 show the frequencies ofpopulation of irradiated molecules, and Ln(0 rad) is thedouble strand breaks per gigabase pair in NotI-cleavednumber average length of the population of unirradi-human DNA irradiated with 1.1 GeV/nucleon Fe26/

(from the gel in Fig. 1). The lower line shows DSBproduction in NotI-cleaved human DNA irradiatedwith 137Cs g rays. Because of the low frequencies ofDSBs, the images of gels containing g-irradiated DNAdo not show such a visually striking increase in smallermolecular length fragments within the 1.2- to 1.3-MbpNotI cleavage group; however, the number average mo-lecular lengths of this group decreased monotonicallyfor DNAs subjected to increasing irradiation. For exam-ple, for one TAFE gel containing DNAs irradiated with0, 2, 5, 10, 50, and 100 cGy of g rays, the numberaverage molecular lengths were 1350, 1338, 1314,1298, 1265, and 1175 kbp, respectively. However, atdosesúÇ50 cGy, radiation-induced scissions recruitedadditional DNAs from the well area, making uniqueidentification of the high molecular length extent of the1.2- to 1.3-Mbp NotI group difficult. The data show thatFIG. 2. DNA dispersion function constructed from the molecularthe assay is linear up to at least 30 DSB/Gbp.length (L) vs migration position (x) calculated from the S. cerevisiae

and H. wingei chromosomes and l concatamers (j). The data for DSB induction by Fe26/ show clear in-



FIG. 3. DNA profiles from NotI-digested human DNA, either unirradiated (solid line) or irradiated with 4 cGy of Fe26/ ions (dotted line),corresponding to lanes 1 and 3, respectively, of Fig. 1. The limits of integration for determination of number average molecular length werepixels 143–210 for lane 1 (unirradiated) and 144–244 for lane 3 (4 cGy).

creases with dose (see Fig. 1); however, the doses used diation buffer. Using a ratio of 70 for DSB inductionin isolated DNA irradiated in a phosphate buffer in[chosen on the basis of the expectation of an equal or

lower yield of DSB per unit dose of iron ions of this the absence of radical scavengers such as Tris orEDTA vs the same DNA in cells (26, 41, 53), thisenergy relative to g rays (18, 29)], produced a higher

level of DSB than did the g rays. Due to the availability value corresponds to Ç4.6 DSB/Gbp/Gy in cells. Thisnumber can be compared with values obtained fromof the heavy ion source for a limited time only once per

year for biological experiments, it was not feasible to cells irradiated at higher doses and analyzed usingdifferent approaches: neutral sedimentation, 2.6– 8carry out additional experiments using lower doses.

These data also illustrate a second type of interference DSB/Gpb/Gy (24, 25); neutral elution, 4– 8 DSB/Gbp/Gy (1, 34); and various pulsed field regimes, 4.6– 6.6with measurement at higher DSB frequencies: DNA

scission resulting in reduction in size of molecules com- DSB/Gpb/Gy (1, 4, 19).Variable results have been obtained by different in-prising the 1.2- to 1.3-Mbp NotI cleavage group pro-

duces fragments that migrate between the 1.2- to 1.3- vestigators using a variety of approaches for the fre-quencies of DSB produced per dose of heavy ions ofMbp group and the group of smaller fragments cen-

tered at about 350 kbp. This obscures the true extent comparable energy and linear energy transfer value(18, 20, 29). Our data clearly indicate higher yields ofof the ‘‘tail’’ of DNAs at the small molecular length edge

of the 1.3-Mbp group, producing an underestimate of DSBs per dose induced by Fe26/ than by g rays, morein agreement with the data of Kampf (20), who usedthe number of these small molecules in the group. This

results in an overestimate of the number average mo- sucrose gradient sedimentation to measure DSB fre-quencies, than with those of Lobrich et al., who mea-lecular length, and thus an underestimate of the fre-

quency of double strand breaks. Thus, we limit the sured depletion of a radioactively probed NotI cleavagepopulation on CHEF gels (29), or of Heilmann et al.,range of the current assay, for human DNA cleaved

with NotI and electrophoresed using this TAFE regime, who used neutral elution (18).to Ç30 DSB/Gbp.

The yields of DSB induced by g rays measured byDISCUSSION

this method agree with those obtained by other meth-ods: the data in Fig. 3 correspond to a DSB induction Calculation of the number average molecular lengths

by the method of moments of DNAs in a population isyield of Ç3.2 DSB/Gbp/cGy. The frequency of strandbreaks induced in isolated DNA depends on the irra- a general method for determining DNA strand break



trophoretic gels. There are several pulsed-field electro-phoretic modes and many regimes of agarose and buffercomposition, field strength, and voltage pulsing sched-ules. We tested contour-clamped homogeneous electricfield electrophoresis, which allows very large moleculesto enter the gel, but shows severe compression in somemolecular length ranges (28, 31, 32, 55); it did not ap-pear to be useful for determining DSBs at low breakfrequencies. However, transverse alternating field elec-trophoresis (15, 16) with an appropriate pulsing regimeproduces straight and colinear lanes (Fig. 1) and re-solves DNAs in the range Ç50–1600 kbp (see Fig. 2).

Determining the number average length of a DNApopulation requires measuring both the lengths andnumber of molecules of each length in the populationof interest. Thus, accurate quantitation of DNA mole-

FIG. 4. The frequency of double strand breaks obtained using num- cules in the various size classes is essential. Such quan-ber average length calculations for production of double strand titation requires both quantitative gel methods (high-breaks (per Gbp) as a function of dose of 137Cs g rays to NotI-cleaved quality agarose gels of uniform thickness and composi-human DNA (m, ., data from two independent gels). After electro-

tion; uniform ethidium staining and destaining; avoid-phoresis using TAFE, the gel was stained with ethidium and de-ance of accumulation of particles from water, dust, orstained and a quantitative electronic image was obtained. A disper-

sion curve was constructed using the yeast chromosomal DNA bands powdered gloves; use of high-purity water in gel andand l concatamers as molecular length standards, and the number buffer; control of electrophoretic temperature) andaverage molecular lengths were calculated according to Eq. [1]. From quantitative recording of DNA mass. For nonradioac-the number average molecular lengths of the unirradiated and each

tive DNA, fluorescence from DNA-bound ethidium canirradiated DNA, the frequency of double strand breaks in each sam-be recorded by photographic film, but film has a limitedple was calculated from Eq. [2]. The straight line was fit to the

experimental data by the method of linear least squares. The slope dynamic range and nonlinear response. We used a sec-of this line is 3.2 DSB/Gbp/cGy. Values for DNA irradiated with 1.1 ond generation version of a CCD-based system withGeV/nucleon Fe26/ ions (n, s) are also shown. a uniform transilluminator and minimized vignetting

that gives a linear response to fluorescence from ethid-ium bromide bound to DNA (46).

In our original development of this approach forfrequencies. It is based on properties of the DNA mole-cules rather than empirical considerations, does not quantitation of DNA lesions using agarose gels (14), all

the DNA in the sample was included in the calculation.require the assumption that the distribution of breaksin a population is random, and offers the potential of However, the method of moments analysis also can be

applied to any subpopulation of DNA in the sample ashigh sensitivity. Application of this method to quantita-tion of double strand breaks at low frequencies (e.g., at long as there is a method of selecting that subpopula-

tion for analysis. The protocol involving digestion oflow doses of ionizing radiation corresponding to highbiological survival) requires that four criteria be met: human DNA by NotI followed by TAFE provides a sepa-

ration into three distinct subpopulations, one of whichfirst, high-molecular-length DNA must be obtainedfrom the cells (and DNAs of known sizes spanning the (the group centered at Ç1.2–1.3 Mbp) is suitable for

high-sensitivity lesion quantitation.range of the experimental DNAs must be available aslength standards); second, (groups of) fragments must The slope of the dispersion function increases for mo-

lecular lengths greater than about 1.1 Mbp, corre-be obtained from the DNA that are still large enoughfor high sensitivity but small enough to migrate in an sponding to a reduction in electrophoretic resolution.

However, measurements of DNA mass per band in S.electrophoretic gel; third, an electrophoretic mode andregime must be selected that resolves such fragments cerevisiae DNA indicates that the slowest-moving band

contains DNA from the 1.6-Mbp chromosome, with noaccording to their molecular lengths and producesstraight, parallel lanes; and fourth, the size and mass significant contribution from the 2.2-Mbp chromosome

(data not shown). This implies that the DNA from theof DNAs at pertinent migration positions on the gelmust be quantitated accurately. 2.2-Mbp chromosome does not enter the gel under our

conditions, in agreement with the results of GardinerIsolation of human DNA by digestion of cells embed-ded in high-quality agarose fulfills the first criterion. (15). Thus the 1.2- to 1.3-Mbp groups in the sample

lanes do not contain DNA molecules much larger thanAlthough undigested DNA is too large to enter neutralelectrophoretic gels, NotI gives a convenient set of frag- 1.6 Mbp. While the larger molecules in the 1.2- to 1.3-

Mbp group are less well resolved because of the slopement groups that readily migrate into appropriate elec-



of the dispersion function, they are also less heavily ity of suffering breaks than smaller ones, the 1.2- to1.3-Mbp group will suffer more hits/DNA molecule/doseweighted in the calculation of number average length

because of the presence of 1/L in the integrand of the than the 0.35-Mbp group; thus increasing DSB levelswill produce smaller molecules from the 1.2- to 1.3-Mbpnumerator of Eq. [1].

In principle, the 1.2- to 1.3- and 0.35-Mbp groups group that can approach the larger molecules in theÇ0.35-Mbp group, obscuring the lower molecular limitcould be combined in the analysis. However, since the

mass of DNA in these groups is comparable, the total of the 1.2- to 1.3-Mbp group.The range of frequencies of sites in genomic DNAsnumber of molecules in the combined population would

be dominated by the population of the smaller mole- that can be quantitated by this method depends on thesize and distribution of molecules in the cleavage groupcules. In the method of moments calculation we mea-

sure changes in the number of molecules between irra- generated by a restriction enzyme, and thus on theDNA base composition and sequence, a characteristicdiated and unirradiated populations. The dose required

to produce at least one DSB in a given fraction of the of each species. The choice of a suitable restriction en-zyme for a given species is thus critical: an ideal en-population of the smaller molecules is much greater

than the dose to produce breaks in the same fraction zyme should yield a cleavage group of sufficient num-bers of molecules for easy quantitation, which is largeof the population of larger molecules. Thus, including

the population of smaller molecules would reduce the enough for high sensitivity, but small enough to mi-grate on an electrophoretic gel under optimized condi-sensitivity of the measurement. To examine the effects

of low doses, we focus exclusively on the subpopulation tions and is also easily separated from other groups ofcleavage products. While NotI is satisfactory for humanof larger molecules, i.e., the 1.2- to 1.3-Mbp group.

Strictly speaking, the frequencies of DSB formation DNA, it will not be suitable for all species.This method provides sensitivity in the cGy range,determined by the present method apply only to that

subpopulation of DNA molecules generated by NotI di- corresponding to the production of DSBs up to about30 sites/Gbp. It is also important to be able to measuregestion to which the analysis is applied. However, the

potential number of double strand cleavage sites is very damages at even lower levels. Use of other newly de-scribed rare cutting enzymes and electrophoresislarge, essentially equal to the number of base pairs

in the sample, and substantial evidence indicates that modes that can resolve larger DNA molecules mayallow such measurements.radiation-induced double strand breaks in mammalian

DNA are distributed at random (11, 21). Thus the re-sults of the method of moments analysis presumably ACKNOWLEDGMENTSapply to all the DNA in the irradiated sample. Recent

This research was supported by the Office of Health and Environ-evidence suggests that some DNA lesions, e.g., singlemental Research of the U.S. Department of Energy, by a Nationalstrand breaks, may not be distributed randomly andInstitutes of Health Grant HG00371 to J.C.S., and by the Spacemay be influenced by various intracellular factors (7, Radiation Health Program, Life and Biomedical Sciences and Appli-

10, 26, 27, 30, 49, 54). Bunch et al. used a combined cations Division of the National Aeronautics and Space Administra-tion. We thank R. Sautkulis, Biology, BNL for irradiating samplesalkaline unwinding/Southern blotting method to studywith 137Cs g rays and providing dosimetry, Dr. Jack Miller, Lawrencedifferential damage and rejoining in megabase-sizeBerkeley Laboratory, for providing dosimetric measurements forDNA fragments containing known genes in MCF-7heavy ion irradiation, and Dr. Richard Setlow for critical reading of

breast cancer cells exposed to 2–10 Gy of g rays (7). the manuscript.The combination of our approach of number averagelength analysis of NotI-cleaved human DNA with de-

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Double Strand Breaks Induced by Low Doses of γ Rays or Heavy Ions: Quantitation in Nonradioactive Human DNA