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Integrity of parental DNA during replication
.I. Mol. Biol. (1978)
120, 281-295
Integrity of Parental DNA During Replication DAVID
R. STRAYERt AND PAUL D. BOYER
Department of Chemistry and Mobcular Biology Institute University of California, Los Angeles, Calif 90024, U.S.A (Received 23 August
1977, and in revised form 28 December 1977)
The integrity of parental DNA aa assessed by thymine, phosphate, or phosphodiester bond oxygen replacement was studied during replication of Eacherichia coli 15T-. Cells containing density labeled (13C, 15N) parental DNA were allowed to replicate for one generation in media (‘ZC, 14N) containing [3H]thymine and 32P0,, or C3H]thymine and Hzl*O, or 32P04 and H,180. After DNA isolation, the single-stranded parental strands were purified by equilibrium centrifugation on sequential sodium iodide density gradients. Measurements were made of 3H and 32P in the DNA and of 180 in phosphate derived from the DNA phosphodiester linkages. The amount of 3H and 32P detected in the parental DNA was used to set upper limits on thymine (1 per 10,000 to 20,000 thymine residues) and phosphate (1 per 7000 bases) replacement in each parental DNA strand. The phosphate from parental DNA contained more I80 following replication in ‘*O-enriched Hz0 than before the exposure. This increased I80 content is consistent with frequent (1 per 100 to 500 bases) hydrolytic cleavage of the phosphodiester backbone of each parental DNA strand per round of replication. Such nicks could act as multiple swivels and facilitate unwinding of parental DNA strands during replication and transcription.
1. Introduction The molecule of DNA that makes up the genome of Escherichia c&i is approximately 1.1 mm in length, corresponding to a molecular weight of 2.8 x 10B, and is in the form of a ring. The concept of such a large circular DNA molecule raises some intriguing questions regarding the integrity of such molecules during replication and exposure to repair, transcription and recombination processes. Since preservation of the specific base-pair sequence of DNA is fundamental for the transfer of genetic information, mechanisms might have evolved to minimize the number of nicks or gaps placed in DNA during replication. Alternatively, it may have been more advantageous to accept frequent breakage of the DNA molecule and to insure fidelity by highly efficient repair mechanisms. Although the process of DNA replication and repair has received intensive investigation, the extent of DNA cleavage and repair which occurs during normal replication has not been well-defined. The unwinding of a closed circular DNA molecule during semiconservative replication creates a “swivel problem”. As the parental strands unwind to serve as templates, a torque would be transmitted to the part of the molecule ahead of the growing point. One superhelical turn would be created in the DNA molecule for every ten t Present address: Laboratory of Tumor Room 6B04, Bethesda, Md 20014, U.S.A.
Cell Biology,
National
Cancer Institute,
Building
37,
281 0022-2836/78/1202--8196
$02.00/O
0 1978 Academic
Press Inc. (London)
Ltd.
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D. R. STRAYER
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bases added until further replication
was prevented because of conformational strain (Sebring et al., 1971). The existence of one biological swivel would permit the unwinding of the parental strands. A single-strand break or nick in one phosphodiester linkage of a parental DNA strand could act as such a swivel by allowing free rotation around the other DNA strand. However, since the chromosome of E. coli is replicated at rates up to lo5 base-pairs per minute, a large portion of the chromosome would be required to rotate at rates up to lo4 revs/min (Tomizawa & Ogawa, 1968). A single swivel point seems even less likely, since the replicating E. coli chromosome is membrane-attached and contains 12 to 80 loops of supercoiled DNA folded into a compact structure (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972,1974; Delius & Worcel, 1974). Since a single-strand nick allows relaxation of the superhelical turns within that loop without altering the superhelical content of other loops, it seems likely that at least one nick per loop would be necessary for DNA replication. If hydrolytic cleavage and resynthesis does occur, it is less frequent than one in live residues along the DNA chain (Richards & Boyer, 1966). Experiments were done to study the integrity of DNA during normal replication. Upper limits have been determined for thymine and phosphate replacement in parental DNA. In addition, the findings are consistent with relatively frequent hydrolytic cleavage of the phosphodiester backbone during normal replication.
2. Materials and Methods (a) Bacterial strain E. coli 15T- was a generous gift from Dr Patrice J Zamcnhof, Department Chemistry, University of California, Los Angeles.
of Biological
(b) Isotopic materials [13C]glucose (72 atom y0 13C) and [13C]starch (76 atom y/, 13C) were provided through the generous co-operation of Dr Donald Ott of the Los Alamos Scientific Laboratory of the University of California. 15NH,C1 (99 atom “/ 15N) was obtained from Bio-Rad Laboratories. [3H]thymine was purchased from New England Nuclear. H,laO was purchased from Miles Laboratories or through the Monsanto Research Cooperation, Mound Laboratory, P.O. Box 32, Miamisburg, Ohio 45342. (c) Other materials
Ribonuclease A type IIIA from bovine pancreas was purchased from Sigma Chemical Co. Ethidium bromide was purchased from Calbiochem. Cesium chloride was obtained from Harshaw Chemical Co. Millipore HAWP and GSWP membrane filters were obtained from Millipore Corporation. Highly polymerized salmon sperm DNA was purchased from Mann Research Laboratories, Inc. All other chemicals were reagent grade products and were used without further purification. (d) Culture media Medium P contained per liter: 5 g KH,PO,, 12 g K2HP04, 5 g NaCl, 3 g NH,Cl, 10 g glucose, 0.12 g MgS04, 5 mg FeSO,, and, unless otherwise noted, 3 mg thymine. 15NH4C1 and [13C]glucose (or 13C-labeled, hydrolyzed starch) replaced 14NH4C1 and [12C]glucose when density labeling was desired. Each gram of starch was hydrolyzed in 800 ml of 0.03 M-HCl by heating to 120°C at a pressure of 15 lb/in2 for 4 h in an autoclave. The concentration of thymine was reduced to as low as 0.6 mg/l for [3H]thymine incorporation. Medium T, identical to medium P, except that it contains 12 g Tris, 1 g sodium citrate,
PARENTAL
DNA
INTEGRITY
and 50 to 500 mg KH,PO, per liter (in place of the 5 g KH,PO, used for 32P, (inorganic phosphate) incorporation.
(e) Bacterial
283 and 12 g K,HPC),)
was
growth
The cells were kept on slants which were transferred monthly. Each experiment \vss begull b>- inoculation of a lo-ml starter culture. After approx. 12 II of growth, the starter culture wa,s used to inoculate the final culture. When the final medium contained 15NH,CI and [13C]glucose, the starter culture contained 3 g of both compounds/l instead of 1 g/l. (‘ells were grown in Erlenmeyer flasks on a rotary shaker at 37°C to optical densities of ; 1.5 at 650 nm. When greater optical densities were required during the growth in H2180, dry air was bubbled through dispersion tubes into the medium contained in tt 5-l Y-neck flask at 37°C. Condensers cooled with circulating water at 4°C were used to lninimize the loss of H,l*O by evaporation. Cells were harvested for media changes by centrifugation at 10°C for 10 min at 6000 re\-s/ Inin in a GSA rotor on a Sorvctll RC-2 centrifuge and resuspended in t)he desired medium. Growtjh was followed turbidimetrically by determining the optical densities of portions of the crll cultures by measuring absorbance at 650 nm. If the ABsO was greater than O%O. the portion was diluted with medium t,o an A,,, value between 0.3 and 0.6. (f) Isolation
of DATA
‘1’11(! cells were llarvested from the growth media by centrifugation at 4°C for 10 111111 ;rt, 7000 rers/min in a Sorvall RC-2 centrifuge. The cell pellet was washed by resuspension ill 200 ml of Tris/EDTA (0.1 M-Tris, 0.1 M-EDTA, pH 8.0) and recentrifuged under identical conditions. The washed cells were dispersed in Tris/EDTA at a concentration trf 2.5 x lOlo cells/ml for DNA isolation by the procedure of Marmur (1961). The cells werr Iysed by the addition of 0.1 vol. sodium dodecyl sulfate (20%, w/v) and heating at 60°C fi)r 10 min. After cooling the lysate to room temperature, 0.25 vol. 0.5 ~-N&10, and 1 vol. isoamylalcohol/CHC13 (1: 24, v/v) were added prior to 30 min of shaking on a rotary shaker. After centrifugation, the aqueous layer was removed with a large-bore pipet to minimize I)reekage of the DNA. This deprot,einization step was repeated 2 or 3 times as necessltr)until only 8 minimal amount of material layered at the CHCl,/water interface. The DNA was precipitated and spooled onto glass rods after the addition of 2 vol. ethanol. The spooled strands were dissolved in 0.1 x SSC (SSC is 0.15 M-N&, 0.015 M-Na cit,ratc) with gentle stirring. The DNA was incubated at 37°C with 25 pg RNase/ml for 60 min. The RNase was previously heated at 80°C for 10 min to destroy possible contaminating DNtlse. The deproteinization step was repeated as already described and the DNA again prclcipitated with ethanol. This purified DNA was dissolved in 0.1 > SSC containing 0.001 >I-EDTA (pH 7.5) and stored at 4°C. ‘l’l~c ;tmorlnt of DNA was determined by t)he method of Dischc (1930) using the modification described by Burton (1956). When the amourlt of DNA precipitated onto Millipore filters needed to be determined, a slight modification of the Dische t,est was used. Millipore filters containing the DNA samples and known DNA standards (0 to 70 pg DNA/filter) were placed in individual vials and covered by a mixture of 0.6 ml of 0.25 MHC104 and 1.2 ml of diphenylamine reagent. The absorbance at 600 nm was measured after 24 h incubation at 25’C and DNA det,ermined from a standard curve. (g) Analytical
centr$ugation
Arralyt,ical centrifugation was used to determine the density distribubion of the E. co&i DNA isolated from each growth experiment. Cesium chloride equilibrium bandings of the DNA were performed on a Beckman model E analytical centrifuge (22 h, 44,700 revs/min, 25°C) in an AND rotor. The density of the &Cl solution containing 0.01 MEDTA, 0.01 >I-Tris.Cl(pH 8) was determined from the refractive index (Messelson et al.. 1957). Ultraviolet films were analyzed with a Joyce-Loebel microdensitometer. The ares, under each pea,k was measured t,o determine tile proportion of each DNA species plYSPlIt.
284
D. R. STRAYER
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(h) Prepamtive centrijugation Preparative NaI equilibrium density-gradient centrifugation was used to separate E. coli DNA species by differences in their buoyant density. NaI density gradients give better separations of two DNA species than CsCl gradients in the same centrifugal field (Anet & Strayer, 1969). This is especially apparent when the dye ethidium bromide is used to locate banded DNA. An added bonus is the relatively low cost of NaI compared to CsCl. A stock solution saturated in sodium iodide and sodium sulfite was kept at 25°C. The NaI density gradient solution contained 65 ml saturated NaI stock solution, 5.0 ml of 0.1 M-Tris.Cl-, 0.1 M-EDTA (pH 8-O), 5.0 ml of 1 mg ethidium bromide/ml, and 25 ml of denatured DNA in 0.1 x SSC per 100 ml. The DNA was denatured by heating to 100°C for 20 min, followed by rapid cooling in an ice slurry. The refractive index of the density gradient solution was adjusted to 1.4440 by the addition of a small volume of water before centrifugation in a Ti50 rotor on a Beckman-Spinco model L centrifuge at 10°C for 90 h at 40,000 revs/min. Up to 1 mg of DNA per centrifuge tube was separated by this technique. The ethidium bromide visualized the DNA bands, allowing them to be photographed using illumination from ultraviolet lights. Exposure times of 10 min were required using Kodachrome 25 slide film. Only rarely were tubes photographed and exposure to visible light was minimized. The top DNA band (L strand) was collected through a 24-gauge needle inserted in the side of the polyallomer tube. The residual NaI solution at the bottom of the tube contained the parental DNA (H strand). This DNA was pooled and again denatured by heating to 100°C for 20 min before recentrifugation. (i) 3H and a2P analyses Each DNA sample or gradient fraction was precipitated with 1 ml of 3 M-trichloroacetic acid at 0°C and collected on 0.45 pm Millipore filters. The dried Millipore filters were placed in 10 ml of toluene containing 3.6 g PPO and 90 mg POPOP/l. 3H and 3zP were counted on a Packard Tri-Carb liquid scintillation counter. The filters were washed with toluene and dried before determination of their DNA content. There was no detectable loss of DNA from the filters into the toluene scintillation fluid. (j) I80 analyses
DNA samples were diluted with carrier DNA to obtain enough DNA (1.2 mg) per sample for accurate 180 analysis. DNA samples were degraded by oxidation with alkaline permanganate at 100°C for 4 h to obtain P, by the method of Richards & Bayer (1966). The samples were made 0.12 M in ammonium molybdate and 1.04 M in HCl and the P, was extracted with isobutanol/benzene (1: 1, v/v) and precipitated as MgNH,PO,. The MgNH,P04 was converted to KH,PO, and the oxygens of KHzPO, were converted to CO, for mass spectrometer analyses as described by Richards & Boyer (1965).
3. Results Experiments 1 and 2 were designed to determine the integrity of parental DNA strands as assessed by thymine and phosphate replacement during replication. Cells containing density labeled (13C, 15N) parental DNA were exposed to [3H]thymine and 32P1for one generation. After DNA isolation the radio-labeled daughter strands were separated from the parental strands on the basis of their different buoyant density in gradients of sodium iodide. Purifications greater than lO,OOO-fold were achieved after multiple sequential sodium iodide gradient separations. The amount of 3H and 32P present in the purified parental DNA was measured, and upper limits for thymine and phosphate replacement in parental DNA were determined. Experiments 3 and 4 were designed to detect possible hydrolytic cleavage of the phosphodiester bonds in the parental DNA. In experiment 3 cells containing density labeled and 32P-labeled parental DNA were exposed to [3H]thymine and H,lsO for
PARENTAL
DNA
INTEGRITY
285
one generation. The 32P/3H ratio was used to follow the purification of the parental DNA during the sequential sodium iodide gradients. The measurement of 3H present in the parental DNA set upper limits on thymine replacement as in experiments 1 and 2. The measurement of the ls0 content of the phosphate derived from the parental DNA phosphodiester linkages set limits on the frequency of hydrolytic cleavage. Tn experiment 4 cells containing density labeled and 3H-labeled parental DNA were exposed to 32P1 and H,laO for one generation. The 3H/32P ratio was used to follou t,he purifi&ion of the parental DNA away from phosphate contaminants. Again the measurement of the la0 content of phosphates from parental DNA set limits on the Srequency of hydrolytic cleavage.
(a) Growth patterns and isotope labeling E. co& UT- cells containing 13C, 15N density labeled parental DNA usually grew logarithmically before and after transfer to 12C, 14N media as shown in Figure 1. The growth interval (b) --f (c) was necessary for the synthesis of DNA segments which physically separated the density labeled parental DNA from the native density daughter st’rands. The parental DNA sustained a second isotopic exposure in the absence of any density label for approximately one generation of cell growth (d) + (e). The density distributions of DNA B and DNA F isolated at (c) and (e), respectively. were determined from microdensitometer tracings. A typical tracing is shown in Figure 2. Observed density distributions in the four experiments are summarized in Table 1. The fraction of parental strands (H) which replicated and light strands (L) which were synthesized during the radioisotopic exposure is given in Table 2. Thesc fract’ions were calculated assuming no selective loss of either H strands or L strands L strands are designated during the logarithmic growth from (d) --f (e). Radio-labeled by L*. TABLE
1
Density distribution of DNA isolated from growth experiments Experioment
DNA
1
B F B F B F B F
2 3 4
%LL
%LH
0.0 27.0 0.0 15.1 0.0 3% 1 0.0 24.1
TABLE
70.6 69.1 62.2 71.4 76.9 60.2 61.5 75.9
:/,HH 29.4 3.9 37.8 13.5 23.1 1.7 38.5 0.0
2
Replication of H and L DNA strands during isotopic exposure Experiment IlO.
1 2 3 4
y0 of H strands which replicated 70.4 42.8 92.2 87.4
“/( of L strands that were synthesized 66.9 56.3 70.9 72.8
D.
286
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3.0
AND
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I
-3.0 (el
t
$! 2.0
I-0
0.6
0.6
(b)
Expt
3
(e
0.6
100 Trne
200
303
hn)
Fro. 1. Growth of E. coli 16T- as measured turbidimetrically by optical density at 660 nm during the 4 isotopic density-shift experiments. At (e) the cells were harvested by centrifugation and DNA isolated (DNA F). Eqnerime& 1 Cells growing in 1 1 of medium P containing 1 g hydrolyzed [13C]starch/l and 1 g 15NH,Cl/I were transferred by centrifugation (E) + (b) and resuspended in 1.6 1 of medium T containing 160 mg KHcPOJ. At (c) 0.6 mCi [3H]thymine and 10 mCi saPl were added after 100 ml of medium was removed for DNA isolation (DNA B). Eqneriment 2 Cells growing in 1 1 of medium P containing 1 g hydrolyzed [r3C]starch/l and 1 g r5NH4Cl/l were transferred by centrifugation (a) + (b) and resuspended in 2 1 of medium T containing 60 mg KHsP04/1 and 0.6 mg thymine/l. At (c) 2.6 mCi [3H]thymine and 17.6 mCi 3aPi were added after 160 ml of medium was removed for DNA isolation (DNA B). Eqneriment 3 Cells growing in 3 1 of medium P containing 1 g [W]glucose/l, 1 g ‘6NH,C1/1 and 1.7 mCi 3aPi/l were transferred by centrifugation (a) + (b) and resuspended in 2 1 of medium P. At (0) 100 ml of medium were removed for DNA isolation (DNA B) and the remaining cells were transferred by oentrifugation (c) --f (d) and resuspended in 2 1 of medium P containing 1.6 atom y0 excess Hzl*O, 1.6 mg thymine/l and 3.6 mCi [sH]thymine/l. Expetiment 4 Cells growing in 1 1 of medium P containing 2 g [W]glucose/l, 2 g 15NH,C1/1, 6 mg thymine/l and 0.28 mCi [3H]thymine/l were transferred by centrifugation (a) -+ (b) and resuspended in 1 1 of medium T containing 6 mg thymine/l and 160 mg KHsPOJ. At (a) only 80% of the cells were transferred to medium T, while 20% were used for DNA extraction (DNA A). At (c) 60 ml of medium were removed for DNAisolation (DNAB). At (d) 66 ml of HzlaO (70 atom y. excess) containing 4.6 g NH,Cl and 10 mCi 3aPi were added.
PARENTAL
DNA
INTEGRITY
97
LI-f
LH
LL HH
cc-,
J
;
F DNA
Fro. 2. i\.licrodensitometer
0 DNA
tracings showing the density distribution
of DSA from experiment,
2.
(b) Enrichment of parental DNA The combined 13C, 15N density label permitted good separation of denatured H and L DNA strands on sodium iodide density gradients as noted in Figure 3. Further details on this separation procedure for single-stranded DNA are given elsewhere (Strayer, manuscript in preparation). The parental DNA isolated from one set’ of gradients was repeatedly recombined and banded on additiona’ sodium iodide gradients to obtain highly enriched parental DNA. Table 3 shows the sequential purification of parental DNA in experiment 3. The purification factors presented in Table 3 represent the degree to which H strands in DNA F were separated from radiolabeled L strands (L*). The purification factor was determined in experiments 3 and 4 from the specific activities and initial ratios of these two components of DNA F and the 3H/32P ratio of the enriched parental DNA. The purification factor for experiment 4 was calculated as follows. The ratio of 3H cts/min: 32P cts/min was 1196 for the purified parental DNA used for la0 analyses. The specific activities of L* and H were TABLE 3 Sequential Centrifuge run number
t Purification $ One tube
puri$cution
of parental DNA in experhent
Parental DNA bg/run)
1
2400
2 3 4
1240 640 -
5 6
130
7 8
-
input
55
factor for fraction 0 (Fig. 5). lost in a centrifuge accident
WCLS
Purification factor
Tubes per run
-
12 6 6 4 3 .>
GO 1120 -
2 2&+
4276 13,zoot
during an attempt
3
to reband.
288
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FIQ. 3. Photograph of a centrifuge tube showing the separation of L and H strands of singlestranded DNA F from expt 4. The H band appears to contain more DNA than the L band because of its proximity to the U.V. light source below the tube. A needle was inserted above the H band to remove the L band. The H bands were combined and rerun in NaI gradients repeatedly until the desired purification was obtained. The H strands of DNA F from all experiments were purified in this manner. The light region at the bottom of the tube was produced by reflected light from the ultraviolet light source below the tube.
1250 cts/min 32P and 1410 cts/min 3H per microgram of DNA, respectively. Therefore, the ratio of H DNA/L* DNA was 1060. The composition of DNA F was 38% H DNA and 62% L DNA (Table 1). Only 723% of L DNA was synthesized during the 32P, and H2180 exposure (Table 2). Therefore, the ratio of H DNA/L* DNA in DNA F was 0.842. The purification factor = 1060/0+342=1260. The parental DNA was not radio-labeled in experiments 1 and 2 to enable study of the replacement of both the thymine and phosphate moieties. The purification of parental DNA in experiments 1 and 2 was not followed sequentially but was determined for the DNA fractionated on the final density gradient. (c) Thymine
and phosphate replacement
in parental
DNA
The final purified parental DNA after banding was separated into fractions and the 3H and 32P content of each fraction determined. Results of such bandings and isotope determinations are given in Figures 4 and 5. The lowest residual 3H and 32P detected in the parental DNA (fractions of greatest purification) from the final sodium iodide gradient and the specific activity of 3H (615 cts/min per pg) and 32P (3750 cts/min per pg) in the L* DNA were used to set
PARENTAL
DNA
INTEGRITY
-c -4 -e - I: - It -2 9 Fractton
number
FIG. 4. Upper limit of thymine and phosphate replacement in each parental DNA strand during one generation of growth (expt 1) : NaI density-gradient fractions of the purified parental DNA were drop collected (40 drops/fraction) through a 24-gauge needle inserted into the bottom of thrk polyallomer tube. This density gradient represents the 6th sequential banding in NaI for this parental DNA. Each fraction was heated at 100°C for 20 min after the addition of 3.0 ml of 0.111NsOH and precipitated at 0°C with trichloroacetic acid and collected: onto Millipore filters. Thv alkali treatment was used to minimize any possible RNA contamination. Each filter was washed 3 times with 10 ml of ice-cold 0.03 M-trichloroacetic acid before drying and measurement of 3H and 32P per fraction. The DNA content of each filter was measured by the diphenylamine reactirtlr as described in Materials and Methods. -e-e--, DNA; ---C-o--, thymine; --G-o---, phosphate.
Frochon
number
FIG. 6. Upper limit of thymine and phosphate replacement in each parental DNA strand during 1 generation of growth in expt 2: NaI density-gradient fractions of banded parental DNA were drop collected (25 drops/fraction) and analyzed as in expt 1 (Fig. 4). This density gradient represents the 7th sequential banding in NaI for this parental DNA. -@-a--, DNA; -m-O--, thymine; -O-O-, phosphate,.
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the upper limits on the thymine (1 per 20,000 thymine residues) and phosphate (1 per 6600 bases) replacement per strand in parental DNA during normal replication in experiment 1 (Fig. 4). Enrichment of parental DNA decreased at lower densities, consistent with an increasing contribution from the L* DNA strand. In experiment 2 (Fig. 5) the specific activity of 3H (110,400 cts/min per pg) and 32P (29,600 cts/min per pg) in L * DNA set the upper replacement limits in each parental DNA strand at 1 per 10,000 thymine residues and 1 per 7000 bases, respectively. The higher upper limits for phosphate replacement compared to thymine replacement in these two experiments may reflect the larger number of cellular components which contain phosphate and could be sources of trace contamination. (d) Ia0 replacement in parental DNA Experiments 3 and 4 were designed to detect possible hydrolytic cleavage of the phosphodiester backbone of parental DNA that might occur more frequently than thymine or phosphate replacement. The cells containing 13C, 15N-labeled DNA were exposed to H, Ia0 for approximately one generation, Hydrolytic cleavage and reformation of the phosphodiester backbone by Ha la0 during this exposure could permit the incorporation of Ia0 into the phosphate moiety of the parental DNA. Detection of an increased level of Ia0 would place limits on the frequency of such hydrolytic cleavage. In experiment 3 DNA F was purified in two separate series of sodium iodide gradients. Table 3 follows the 13,200-fold purification of 7.4 mg of DNA F through eight sequential centrifuge runs. The specific activity of DNA L* and DNA H was 80,400 cts/min 3H per pg and 40.8 cts/min 32P per pg, respectively. Analysis of one tube from the eighth centrifugation is shown in Figure 6. The upper limit for thymine replacement was 1 per 8700 thymine residues. The remainder of DNA F (14 mg)
Frctctkm number FIG. 6. Upper limit of thymine replacement per thymine residue in parental DNA during 1 generation of growth in expt 3: NeI density-gradient fractions of the purified parental DNA were drop collected (30 drops/fraction) and analyzed as in experiments 1 and 2. -O-O--, DNA; - q - q -, thymine.
PARENTAL
DNA
INTEGRITY
291
was purified 620-fold in a second series of sodium iodide gradients with recovery of 720 pg of purified parental DNA. The ratio of 3H cts/min : 3aP cts/min in this purified parental DNA was 4.81. The I*0 content of this purified parental DNA (DNA H) is shown in Table 4. There was no significant difference between the ls0 content of the E. co& carrier DNA and DNA A and B. However, the Ia0 content of the parental DNA was significantly greater than that of the carrier and control DNAs (p <0.02). The parental DNA contained 0.2152 atom y0 180 after correction for carrier dilution. The 3H radioactivity detected in this purified parental DNA set the L* strand contamination at a maximum of 0.24%. The L* DNA was synthesized during the H,180 exposure of the parental DNA in media P which contained 15 atom y0 excess H,180. Since only the branch phosphate oxygens are derived from media phosphates. which rapidly exchange oxygens with media H,O, the L* DNA phosphates contained 0.75 atom O/oexcess I80 (Richards & Boyer, 1966). Therefore, the contribution by L* to the l”b content of the parental DNA was only about 0*0018 atom %. The actual la0 content of the parental DNA after the H,lsO exposure was 0.2134 atom s,, or 0.0105 atom 7; greater than before the exposure. This difference is consistent with one hydrolytic cleavage per 150 bases per strand if all the oxygens which enter the DNA phosphate at the time of cleavage are retained upon resynthesis of the phosphodiester bond. If, however, as seems likely the three phosphoryl group oxygens become equivalent, the entering oxygen has only a 213 chance of remaining in the DNA with reversal of the hydrolytic reaction and closure of the nick in the phosphodiester backbone. In this case, the frequency of hydrolytic cleavage per strand would be I per 100 bases or greater than @I-fold more frequent than the upper limit for thymine replacement. The increased la0 content detected in the parental DNA was not secondary t&oan elevated ls0 content in the [13C]glucose used to density label the parental Dn’A, since the l*O content of the [13C]glucose was not statistically different from the la0 cement of normal abundance glucose. In addition, control DNA grown at’ the same time in the same ]13C]glucose media as the parental DNA, but not exposed to the H,180, had no increase in Ia0 content. TABLE 4
IlNA source
Atom 4’ ,” 180
Average atom SC,
DNA (rg)
Carrier (NT)
DNA B DNA A
275 276
1100 1100
0.2040 0.2017
0.2029
DNA H (parental)
360
1070
0.2066 0.2065
0.2065
-
2300
E. coli carrier DNA
0.2022 0.2046 0.2041
0.2036
Each DNA sample was diluted with E. coli DNA (carrier) to obtain a total of approx. 1400 pg of DNA per sample for IsO analysis. Each figure in the atom Oc / IsO column represents a simultaneous but independent work-up of each DNA sample from the alkaline permanganate oxidation step through mass spectrometrio analysis of its IsO content. DNA B was extracted from cells removed at (c) Fig. 1 (expt 3) just before the Hz I80 exposure. DNA A was extracted from E. coli lST- cells grown in 11 of medium P. DNA H was the enriched parent)al DNA.
292
D.
R.
STRAYER
AND
P.
D.
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Contamination of the purified parental DNA by non-DNA phosphate compounds containing ls0 was unlikely because of the purification procedures used. For example, these procedures should separate DNA from any RNA, particularly since any RNA present would be hydrolyzed by treatment with 0.1 M-NaCH at 100°C for 20 minutes before trichloroacetic acid precipitation of the DNA onto Millipore filters. Nonetheless, a more positive control on possible 180-phosphate contamination was felt necessary. Experiment 4 was performed under conditions that allowed critical evaluation of possible contamination of the parental DNA by phosphates containing 180, since the H,180 exposure of the parental DNA was done in the presence of 32P,. The parental DNA (14 mg) was purified 1260-fold away from 32P-containing materials with a recovery of 1.31 mg of purified parental DNA (see section (b), above, for calculation of purification factor). Medium P contained 4.6 atom o/o excess H2180. The ratio of 3H cts/min: 32P cts/min in the purified parental DNA was 1196. The atom o/o ls0 contents of DNAs in experiments 3 (Table 4) and 4 (Table 5) are not directly comparable, since (1) experiments 3 and 4 were significantly different in design, (2) the H2180 content of medium P was threefold higher in experiment 4 and (3) different carrier DNAs were used in each experiment. The 180 content of parental DNA from experiment 4 is seen in Table 5. The 180 content of the non-isotopic control DNA (DNA A) was not significantly different from the calf thymus carrier DNA. The 180 content of the parental DNA was significantly greater than DNA A (p < 0.005). The 180 content of the parental DNA after correction for carrier dilution and L* strand contamination was 0.2077 atom y. or 0.0052 atom y. greater than DNA A. Assuming three equivalent phosphoryl group oxygens, this difference is consistent with one phosphodiester bond cleavage per 520 bases per round of chromosome replication in each parental DNA strand. TABLE 5 180 content of purified parental DNA in experiment 4 DNA
DNA
SOUITX
DNA
(P?4
Carrier hi9
Atom y0 160
A
215
960
DNA H (parental)
440
720
0.2060 0.2052 0.2056
0.2052
-
1200
0.2027 0.2022 0.2027
0.2025
Thymus carrier DNA
0.2026 0.2017
Average atom o/0 0.2022
Each DNA sample was diluted with calf thymus DNA (carrier) to obtain a total of approx. 1200 pg of DNA per sample for I80 analysis. Each figure in the atom o/o IsO column was determined independently as in Table 4. DNA A was extracted from cells removed at (a) Fig. 1 (expt 4). DNA H was the enriched parental DNA.
4. Discussion These experiments place upper limits on the incorporation of thymine (1 per 10,000 to 20,000 thymine residues) and phosphate (1 per 7000 bases) into each parental E. coli DNA strand during normal replication. The upper limit found for
PARENTAL
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INTEGRITY
293
thymine replacement was lower than that for phosphate replacement. The incorporation rate found for thymine into parental DNA is less than or equal to 50 to 100 thymine residues replaced per DNA strand in the E. coli genome. The rate of incorporation of the three other bases into parental DNA was not studied. The discrepancy between the thymine and phosphate replacement could possibly reflect an increased frequency of incorporation of one or more of the other bases compared to thymine. A more likely explanation is the failure to purify the parental DNA away from nonthymine containing phosphate compounds. These limits reached for phosphate and thymine replacement are, as far as we are aware? the lowest yet observed, approaching one to three per Worcel loop. The increased level of 180 detected in the phosphodiester branch oxygens of parental DNA after replication in media containing H,O enriched in 180 is consistent with hydrolytic cleavage of the phosphodiester bonds (1 per 100 to 500 bases per parental strand). The increased parental DNA l*O content cannot be explained by endonuclease activity during the DNA isolation procedure prior to separation from H,laO. Denaturation of such frequently nicked parental DNA would yield low molecular weight fragments incompatible with the DNA bands obtained (Fig. 3) and t’he enrichment of parental DNA achieved on sodium iodide gradients. Therefore, the majority of the phosphodiester bond cleavages detected must have been resealed prior to DNA isolation. Although the molecular mechanism of the cleavage and resynthesis of the phosphodiester bonds is unknown, a DNA relaxation protein like w (Wang, 1971) could play a possible role in DNA replication. w is a protein found in E. coli that acts like a .‘swivel” removing negative superhelical turns from closed circular DNA in a stepwise manner. Unwinding of DNA during replication produces positive superhelical turns. The failure of Wang to detect relaxation of positive superhelical turns in but does not eliminate w as a bacteriophage lambda DNA by w is bothersome, possible swivel for DNA replication in E. coli. DNA relaxation proteins have also been found in mouse cells (Champoux & Dulbecco, 1972; Vosberg et al., 1974). in eggs of Drosophilia melanogaster (Baase & Wang, 1974) and in human tissue culture cells (Vosberg et al., 1974; Keller, 1975). The unwinding is probably accomplished by a rapid nicking and resealing mechanism that appears to proceed without a’ requirement for external energy (Wang, 1971). Wang has proposed a covalent81y linked protein-DNA intermediate which stores the phosphodiester bond energy during relaxation. The reaction is reversible and the phosphodiester bond is restored as w leaves. Such a mechanism would not be expected to give an increased la0 content in parental DNA. DNA gyrase, an enzymatic activity isolated from E. co&, introduces negative superhelical turns into covalently closed circular DNA in an ATP-dependent reaction (Gellert, Mizuuchi et al., 1976) and appears to be essential for the replication of circular DNA in E. coli (Gellert, O’Deaet al., 1976). Recentevidencesuggests that DNA gyrase consists of two components : (1) a nalidixic acid-sensitive nicking/closing activity (ATP independent), and (2) a coumermycin-sensitive supercoiling activity (BTP dependent) (Sugino et al., 1977 ; Gellert et al., 1977). The nicking/closing activity is distinct from u protein and can relax both negatively and positively supercoiled DNA. DNA gyrase, like w protein, may utilize a covalently linked protein-DNA intermediate that is competent for resealing (Sugino et al., 1977 ; Gellert et al., 1977). However. the cxist’ence of such intermediates for DNA gyrase and w prot,ein remains
294
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R. STRAYER
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tentative. It is possible that energy is stored by an enzyme molecule which has been “activated” by the cleavage reaction to a higher energy state (Wang, 1971). Conformational or structural features in the enzyme protein could favor resynthesis of the phosphodiester bond. For example, the formation of an acyl phosphate from P, without phosphorolysis of a pre-existing covalent bond has been reported by this laboratory (Dahms et al., 1973). The formation of a non-covalent “activated” DNA gyrase or w protein-DNA intermediate could result in the increased ls0 content we observed in parental DNA. A second mechanism which could explain the increased la0 content of parental DNA would utilize an endonuclease and ligase activity (Sebring et al., 1971). DNA replication would proceed with formation of positive superhelical turns until the parental strands could not unwind any further. At this point replication would temporarily cease. An endonuclease, possibly recognizing a strained region of the DNA molecule, could introduce a hydrolytic cleavage that would allow relaxation of the strain and replication could proceed. An alternative possibility would be that endonucleolytic cleavage and ligase repair occur frequently along the length of the DNA molecule and thus prevent conformational strain and cessation of DNA replication. This model would predict an accumulation of single-strand cleavages in DNA with cellular ATP depletion consistent with the results reported by Hilton t Walker (1977). Single-strand breaks could also facilitate the local separation of DNA strands, which occurs during selective gene transcription (Frenster, 1976). Thus, it is possible that events accompanying transcription (Riva et al., 1970) or other metabolic processes could be responsible for some or most of the la0 incorporation we have observed. The authors are very grateful to Dr Ragini Anet for many helpful discussions and for her fine experimental efforts that helped in initiation of these experiments many years ago. Thanks are also due to MS Kerstin Stempel for careful lsO analysis. This work was supported in part by contract E(O4-3)-34) of the Atomic Energy Commission, U.S. Energy Research and Development Administration.
REFERENCES Anot, R. & Strayer, D. R. (1969). Biochem. Biophys. Re.s. Commurr. 37, 52-58. Baase, W. A. & Wang, J. C. (1974). Biochemistry, 13, 4299-4303. Burton, K. (1956). Biochem. J. 62, 315-323. Champoux, J. J. & Dulbecco, R. (1972). Proc. Nat. Acad. Sd., U.S.A. 69, 143-146. Dahms, A. S., Kanazawa, T. & Boyer, P. D. (1973). J. Biol. Chem. 248, 6592-6595. Delius, H. & Worcel, A. (1974). J. Mol. Biol. 82, 107-109. Dische, Z. (1930). Mikrochemie, 8, 4-32. Frenster, J. H. (1976). Cancer Res. 36, 3394-3398. Gellert, M., O’Dea. M. H., Itoh, T. & Tomizawa, J. (l!J76). Proc. iliat. Acad. Xci., U.S.A. 73, 4474-4478. Gellert, M., Mizuuchi, K., O’Dea, M. H., ltoh, T. & Tomizawa, J. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 4772-4776. Gellert, M., Mizuuchi, K., O’Dea, M. H. & Nash, H. A. (1976). I’roc. Nat. Acad. Sci., U.S.A. 73, 3872-3876. Hilton, J. & Walker, M. D. (1977). Biochem. Biophys. Res. Commun.. 77, 9099914. Keller, W. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 2550-2554. Marmur, J. (1961). J. Mol. Biol. 3, 208-218. Messelson, M., Stahl, F. W. & Vinograd, ,J. (1957). Proc. Nat. Acad. Xci., U.S.A. 43, 581-588. Richards, 0. C. & Boyer, P. D. (1965). J. ,Vol. Biol. 11, 327-340. Richards, 0. C. & Boyer, P. D. (1966). J. MoZ. BioZ. 19, 1099119.
PARENTAL
DNA
INTEGRITY
“!I5
Kiva, S., Cascino, A. & Gciduschek, E. P. (1970). .J. Mo7. J3io7. 54, IO3 I I!). Sebring, E. D., Kelly, T. tJ., Thoren, M. M. CycSalzman~ X. I’. (1971). .I. lyirol. 8, 4i8 490. Stoningt~on, 0. C. C%P&ijohn, D. E. (1951). I’roc. Nat. ilcntl. ,C&, 1,T.S.A. 68, 6-9. Srlgino, -4., Peeblcs, (:. L., Krewrr, K. N. & C!oxzarelli, N. Ii. (1977). Proc~. n’at. rlrad. Sci., U.S.A. 74, 4767-4771. Tornizawa. J. & Ogawa, T. (1968). C’old 8prin.g Harbor ~Symp. Quant. Bid. 33, 533. 551. Vosbrrg, H. I’.. Grossman, L. I. & Vinograd. .J. (1974). Ferl. J’roc. Fed. a+imv-. SW. ET/J. Rid. 33, 1356. Weang, ,J. C!. (I 971). .I. Mol. Bid. 55, 523-533. IVorcel, A. & Burpi, E. (1972). J. Mol. Bid. 71, 1277147. \~~orcr:l. A. & Hlugi,
E. (1974). .I. Mol.
Biol.
82, 91- 105.
120, 281-295
Integrity of Parental DNA During Replication DAVID
R. STRAYERt AND PAUL D. BOYER
Department of Chemistry and Mobcular Biology Institute University of California, Los Angeles, Calif 90024, U.S.A (Received 23 August
1977, and in revised form 28 December 1977)
The integrity of parental DNA aa assessed by thymine, phosphate, or phosphodiester bond oxygen replacement was studied during replication of Eacherichia coli 15T-. Cells containing density labeled (13C, 15N) parental DNA were allowed to replicate for one generation in media (‘ZC, 14N) containing [3H]thymine and 32P0,, or C3H]thymine and Hzl*O, or 32P04 and H,180. After DNA isolation, the single-stranded parental strands were purified by equilibrium centrifugation on sequential sodium iodide density gradients. Measurements were made of 3H and 32P in the DNA and of 180 in phosphate derived from the DNA phosphodiester linkages. The amount of 3H and 32P detected in the parental DNA was used to set upper limits on thymine (1 per 10,000 to 20,000 thymine residues) and phosphate (1 per 7000 bases) replacement in each parental DNA strand. The phosphate from parental DNA contained more I80 following replication in ‘*O-enriched Hz0 than before the exposure. This increased I80 content is consistent with frequent (1 per 100 to 500 bases) hydrolytic cleavage of the phosphodiester backbone of each parental DNA strand per round of replication. Such nicks could act as multiple swivels and facilitate unwinding of parental DNA strands during replication and transcription.
1. Introduction The molecule of DNA that makes up the genome of Escherichia c&i is approximately 1.1 mm in length, corresponding to a molecular weight of 2.8 x 10B, and is in the form of a ring. The concept of such a large circular DNA molecule raises some intriguing questions regarding the integrity of such molecules during replication and exposure to repair, transcription and recombination processes. Since preservation of the specific base-pair sequence of DNA is fundamental for the transfer of genetic information, mechanisms might have evolved to minimize the number of nicks or gaps placed in DNA during replication. Alternatively, it may have been more advantageous to accept frequent breakage of the DNA molecule and to insure fidelity by highly efficient repair mechanisms. Although the process of DNA replication and repair has received intensive investigation, the extent of DNA cleavage and repair which occurs during normal replication has not been well-defined. The unwinding of a closed circular DNA molecule during semiconservative replication creates a “swivel problem”. As the parental strands unwind to serve as templates, a torque would be transmitted to the part of the molecule ahead of the growing point. One superhelical turn would be created in the DNA molecule for every ten t Present address: Laboratory of Tumor Room 6B04, Bethesda, Md 20014, U.S.A.
Cell Biology,
National
Cancer Institute,
Building
37,
281 0022-2836/78/1202--8196
$02.00/O
0 1978 Academic
Press Inc. (London)
Ltd.
282
D. R. STRAYER
AND
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bases added until further replication
was prevented because of conformational strain (Sebring et al., 1971). The existence of one biological swivel would permit the unwinding of the parental strands. A single-strand break or nick in one phosphodiester linkage of a parental DNA strand could act as such a swivel by allowing free rotation around the other DNA strand. However, since the chromosome of E. coli is replicated at rates up to lo5 base-pairs per minute, a large portion of the chromosome would be required to rotate at rates up to lo4 revs/min (Tomizawa & Ogawa, 1968). A single swivel point seems even less likely, since the replicating E. coli chromosome is membrane-attached and contains 12 to 80 loops of supercoiled DNA folded into a compact structure (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972,1974; Delius & Worcel, 1974). Since a single-strand nick allows relaxation of the superhelical turns within that loop without altering the superhelical content of other loops, it seems likely that at least one nick per loop would be necessary for DNA replication. If hydrolytic cleavage and resynthesis does occur, it is less frequent than one in live residues along the DNA chain (Richards & Boyer, 1966). Experiments were done to study the integrity of DNA during normal replication. Upper limits have been determined for thymine and phosphate replacement in parental DNA. In addition, the findings are consistent with relatively frequent hydrolytic cleavage of the phosphodiester backbone during normal replication.
2. Materials and Methods (a) Bacterial strain E. coli 15T- was a generous gift from Dr Patrice J Zamcnhof, Department Chemistry, University of California, Los Angeles.
of Biological
(b) Isotopic materials [13C]glucose (72 atom y0 13C) and [13C]starch (76 atom y/, 13C) were provided through the generous co-operation of Dr Donald Ott of the Los Alamos Scientific Laboratory of the University of California. 15NH,C1 (99 atom “/ 15N) was obtained from Bio-Rad Laboratories. [3H]thymine was purchased from New England Nuclear. H,laO was purchased from Miles Laboratories or through the Monsanto Research Cooperation, Mound Laboratory, P.O. Box 32, Miamisburg, Ohio 45342. (c) Other materials
Ribonuclease A type IIIA from bovine pancreas was purchased from Sigma Chemical Co. Ethidium bromide was purchased from Calbiochem. Cesium chloride was obtained from Harshaw Chemical Co. Millipore HAWP and GSWP membrane filters were obtained from Millipore Corporation. Highly polymerized salmon sperm DNA was purchased from Mann Research Laboratories, Inc. All other chemicals were reagent grade products and were used without further purification. (d) Culture media Medium P contained per liter: 5 g KH,PO,, 12 g K2HP04, 5 g NaCl, 3 g NH,Cl, 10 g glucose, 0.12 g MgS04, 5 mg FeSO,, and, unless otherwise noted, 3 mg thymine. 15NH4C1 and [13C]glucose (or 13C-labeled, hydrolyzed starch) replaced 14NH4C1 and [12C]glucose when density labeling was desired. Each gram of starch was hydrolyzed in 800 ml of 0.03 M-HCl by heating to 120°C at a pressure of 15 lb/in2 for 4 h in an autoclave. The concentration of thymine was reduced to as low as 0.6 mg/l for [3H]thymine incorporation. Medium T, identical to medium P, except that it contains 12 g Tris, 1 g sodium citrate,
PARENTAL
DNA
INTEGRITY
and 50 to 500 mg KH,PO, per liter (in place of the 5 g KH,PO, used for 32P, (inorganic phosphate) incorporation.
(e) Bacterial
283 and 12 g K,HPC),)
was
growth
The cells were kept on slants which were transferred monthly. Each experiment \vss begull b>- inoculation of a lo-ml starter culture. After approx. 12 II of growth, the starter culture wa,s used to inoculate the final culture. When the final medium contained 15NH,CI and [13C]glucose, the starter culture contained 3 g of both compounds/l instead of 1 g/l. (‘ells were grown in Erlenmeyer flasks on a rotary shaker at 37°C to optical densities of ; 1.5 at 650 nm. When greater optical densities were required during the growth in H2180, dry air was bubbled through dispersion tubes into the medium contained in tt 5-l Y-neck flask at 37°C. Condensers cooled with circulating water at 4°C were used to lninimize the loss of H,l*O by evaporation. Cells were harvested for media changes by centrifugation at 10°C for 10 min at 6000 re\-s/ Inin in a GSA rotor on a Sorvctll RC-2 centrifuge and resuspended in t)he desired medium. Growtjh was followed turbidimetrically by determining the optical densities of portions of the crll cultures by measuring absorbance at 650 nm. If the ABsO was greater than O%O. the portion was diluted with medium t,o an A,,, value between 0.3 and 0.6. (f) Isolation
of DATA
‘1’11(! cells were llarvested from the growth media by centrifugation at 4°C for 10 111111 ;rt, 7000 rers/min in a Sorvall RC-2 centrifuge. The cell pellet was washed by resuspension ill 200 ml of Tris/EDTA (0.1 M-Tris, 0.1 M-EDTA, pH 8.0) and recentrifuged under identical conditions. The washed cells were dispersed in Tris/EDTA at a concentration trf 2.5 x lOlo cells/ml for DNA isolation by the procedure of Marmur (1961). The cells werr Iysed by the addition of 0.1 vol. sodium dodecyl sulfate (20%, w/v) and heating at 60°C fi)r 10 min. After cooling the lysate to room temperature, 0.25 vol. 0.5 ~-N&10, and 1 vol. isoamylalcohol/CHC13 (1: 24, v/v) were added prior to 30 min of shaking on a rotary shaker. After centrifugation, the aqueous layer was removed with a large-bore pipet to minimize I)reekage of the DNA. This deprot,einization step was repeated 2 or 3 times as necessltr)until only 8 minimal amount of material layered at the CHCl,/water interface. The DNA was precipitated and spooled onto glass rods after the addition of 2 vol. ethanol. The spooled strands were dissolved in 0.1 x SSC (SSC is 0.15 M-N&, 0.015 M-Na cit,ratc) with gentle stirring. The DNA was incubated at 37°C with 25 pg RNase/ml for 60 min. The RNase was previously heated at 80°C for 10 min to destroy possible contaminating DNtlse. The deproteinization step was repeated as already described and the DNA again prclcipitated with ethanol. This purified DNA was dissolved in 0.1 > SSC containing 0.001 >I-EDTA (pH 7.5) and stored at 4°C. ‘l’l~c ;tmorlnt of DNA was determined by t)he method of Dischc (1930) using the modification described by Burton (1956). When the amourlt of DNA precipitated onto Millipore filters needed to be determined, a slight modification of the Dische t,est was used. Millipore filters containing the DNA samples and known DNA standards (0 to 70 pg DNA/filter) were placed in individual vials and covered by a mixture of 0.6 ml of 0.25 MHC104 and 1.2 ml of diphenylamine reagent. The absorbance at 600 nm was measured after 24 h incubation at 25’C and DNA det,ermined from a standard curve. (g) Analytical
centr$ugation
Arralyt,ical centrifugation was used to determine the density distribubion of the E. co&i DNA isolated from each growth experiment. Cesium chloride equilibrium bandings of the DNA were performed on a Beckman model E analytical centrifuge (22 h, 44,700 revs/min, 25°C) in an AND rotor. The density of the &Cl solution containing 0.01 MEDTA, 0.01 >I-Tris.Cl(pH 8) was determined from the refractive index (Messelson et al.. 1957). Ultraviolet films were analyzed with a Joyce-Loebel microdensitometer. The ares, under each pea,k was measured t,o determine tile proportion of each DNA species plYSPlIt.
284
D. R. STRAYER
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P. D. BOYER
(h) Prepamtive centrijugation Preparative NaI equilibrium density-gradient centrifugation was used to separate E. coli DNA species by differences in their buoyant density. NaI density gradients give better separations of two DNA species than CsCl gradients in the same centrifugal field (Anet & Strayer, 1969). This is especially apparent when the dye ethidium bromide is used to locate banded DNA. An added bonus is the relatively low cost of NaI compared to CsCl. A stock solution saturated in sodium iodide and sodium sulfite was kept at 25°C. The NaI density gradient solution contained 65 ml saturated NaI stock solution, 5.0 ml of 0.1 M-Tris.Cl-, 0.1 M-EDTA (pH 8-O), 5.0 ml of 1 mg ethidium bromide/ml, and 25 ml of denatured DNA in 0.1 x SSC per 100 ml. The DNA was denatured by heating to 100°C for 20 min, followed by rapid cooling in an ice slurry. The refractive index of the density gradient solution was adjusted to 1.4440 by the addition of a small volume of water before centrifugation in a Ti50 rotor on a Beckman-Spinco model L centrifuge at 10°C for 90 h at 40,000 revs/min. Up to 1 mg of DNA per centrifuge tube was separated by this technique. The ethidium bromide visualized the DNA bands, allowing them to be photographed using illumination from ultraviolet lights. Exposure times of 10 min were required using Kodachrome 25 slide film. Only rarely were tubes photographed and exposure to visible light was minimized. The top DNA band (L strand) was collected through a 24-gauge needle inserted in the side of the polyallomer tube. The residual NaI solution at the bottom of the tube contained the parental DNA (H strand). This DNA was pooled and again denatured by heating to 100°C for 20 min before recentrifugation. (i) 3H and a2P analyses Each DNA sample or gradient fraction was precipitated with 1 ml of 3 M-trichloroacetic acid at 0°C and collected on 0.45 pm Millipore filters. The dried Millipore filters were placed in 10 ml of toluene containing 3.6 g PPO and 90 mg POPOP/l. 3H and 3zP were counted on a Packard Tri-Carb liquid scintillation counter. The filters were washed with toluene and dried before determination of their DNA content. There was no detectable loss of DNA from the filters into the toluene scintillation fluid. (j) I80 analyses
DNA samples were diluted with carrier DNA to obtain enough DNA (1.2 mg) per sample for accurate 180 analysis. DNA samples were degraded by oxidation with alkaline permanganate at 100°C for 4 h to obtain P, by the method of Richards & Bayer (1966). The samples were made 0.12 M in ammonium molybdate and 1.04 M in HCl and the P, was extracted with isobutanol/benzene (1: 1, v/v) and precipitated as MgNH,PO,. The MgNH,P04 was converted to KH,PO, and the oxygens of KHzPO, were converted to CO, for mass spectrometer analyses as described by Richards & Boyer (1965).
3. Results Experiments 1 and 2 were designed to determine the integrity of parental DNA strands as assessed by thymine and phosphate replacement during replication. Cells containing density labeled (13C, 15N) parental DNA were exposed to [3H]thymine and 32P1for one generation. After DNA isolation the radio-labeled daughter strands were separated from the parental strands on the basis of their different buoyant density in gradients of sodium iodide. Purifications greater than lO,OOO-fold were achieved after multiple sequential sodium iodide gradient separations. The amount of 3H and 32P present in the purified parental DNA was measured, and upper limits for thymine and phosphate replacement in parental DNA were determined. Experiments 3 and 4 were designed to detect possible hydrolytic cleavage of the phosphodiester bonds in the parental DNA. In experiment 3 cells containing density labeled and 32P-labeled parental DNA were exposed to [3H]thymine and H,lsO for
PARENTAL
DNA
INTEGRITY
285
one generation. The 32P/3H ratio was used to follow the purification of the parental DNA during the sequential sodium iodide gradients. The measurement of 3H present in the parental DNA set upper limits on thymine replacement as in experiments 1 and 2. The measurement of the ls0 content of the phosphate derived from the parental DNA phosphodiester linkages set limits on the frequency of hydrolytic cleavage. Tn experiment 4 cells containing density labeled and 3H-labeled parental DNA were exposed to 32P1 and H,laO for one generation. The 3H/32P ratio was used to follou t,he purifi&ion of the parental DNA away from phosphate contaminants. Again the measurement of the la0 content of phosphates from parental DNA set limits on the Srequency of hydrolytic cleavage.
(a) Growth patterns and isotope labeling E. co& UT- cells containing 13C, 15N density labeled parental DNA usually grew logarithmically before and after transfer to 12C, 14N media as shown in Figure 1. The growth interval (b) --f (c) was necessary for the synthesis of DNA segments which physically separated the density labeled parental DNA from the native density daughter st’rands. The parental DNA sustained a second isotopic exposure in the absence of any density label for approximately one generation of cell growth (d) + (e). The density distributions of DNA B and DNA F isolated at (c) and (e), respectively. were determined from microdensitometer tracings. A typical tracing is shown in Figure 2. Observed density distributions in the four experiments are summarized in Table 1. The fraction of parental strands (H) which replicated and light strands (L) which were synthesized during the radioisotopic exposure is given in Table 2. Thesc fract’ions were calculated assuming no selective loss of either H strands or L strands L strands are designated during the logarithmic growth from (d) --f (e). Radio-labeled by L*. TABLE
1
Density distribution of DNA isolated from growth experiments Experioment
DNA
1
B F B F B F B F
2 3 4
%LL
%LH
0.0 27.0 0.0 15.1 0.0 3% 1 0.0 24.1
TABLE
70.6 69.1 62.2 71.4 76.9 60.2 61.5 75.9
:/,HH 29.4 3.9 37.8 13.5 23.1 1.7 38.5 0.0
2
Replication of H and L DNA strands during isotopic exposure Experiment IlO.
1 2 3 4
y0 of H strands which replicated 70.4 42.8 92.2 87.4
“/( of L strands that were synthesized 66.9 56.3 70.9 72.8
D.
286
R.
STRAYER
Expt
3.0
AND
P. D. BOYER
I
-3.0 (el
t
$! 2.0
I-0
0.6
0.6
(b)
Expt
3
(e
0.6
100 Trne
200
303
hn)
Fro. 1. Growth of E. coli 16T- as measured turbidimetrically by optical density at 660 nm during the 4 isotopic density-shift experiments. At (e) the cells were harvested by centrifugation and DNA isolated (DNA F). Eqnerime& 1 Cells growing in 1 1 of medium P containing 1 g hydrolyzed [13C]starch/l and 1 g 15NH,Cl/I were transferred by centrifugation (E) + (b) and resuspended in 1.6 1 of medium T containing 160 mg KHcPOJ. At (c) 0.6 mCi [3H]thymine and 10 mCi saPl were added after 100 ml of medium was removed for DNA isolation (DNA B). Eqneriment 2 Cells growing in 1 1 of medium P containing 1 g hydrolyzed [r3C]starch/l and 1 g r5NH4Cl/l were transferred by centrifugation (a) + (b) and resuspended in 2 1 of medium T containing 60 mg KHsP04/1 and 0.6 mg thymine/l. At (c) 2.6 mCi [3H]thymine and 17.6 mCi 3aPi were added after 160 ml of medium was removed for DNA isolation (DNA B). Eqneriment 3 Cells growing in 3 1 of medium P containing 1 g [W]glucose/l, 1 g ‘6NH,C1/1 and 1.7 mCi 3aPi/l were transferred by centrifugation (a) + (b) and resuspended in 2 1 of medium P. At (0) 100 ml of medium were removed for DNA isolation (DNA B) and the remaining cells were transferred by oentrifugation (c) --f (d) and resuspended in 2 1 of medium P containing 1.6 atom y0 excess Hzl*O, 1.6 mg thymine/l and 3.6 mCi [sH]thymine/l. Expetiment 4 Cells growing in 1 1 of medium P containing 2 g [W]glucose/l, 2 g 15NH,C1/1, 6 mg thymine/l and 0.28 mCi [3H]thymine/l were transferred by centrifugation (a) -+ (b) and resuspended in 1 1 of medium T containing 6 mg thymine/l and 160 mg KHsPOJ. At (a) only 80% of the cells were transferred to medium T, while 20% were used for DNA extraction (DNA A). At (c) 60 ml of medium were removed for DNAisolation (DNAB). At (d) 66 ml of HzlaO (70 atom y. excess) containing 4.6 g NH,Cl and 10 mCi 3aPi were added.
PARENTAL
DNA
INTEGRITY
97
LI-f
LH
LL HH
cc-,
J
;
F DNA
Fro. 2. i\.licrodensitometer
0 DNA
tracings showing the density distribution
of DSA from experiment,
2.
(b) Enrichment of parental DNA The combined 13C, 15N density label permitted good separation of denatured H and L DNA strands on sodium iodide density gradients as noted in Figure 3. Further details on this separation procedure for single-stranded DNA are given elsewhere (Strayer, manuscript in preparation). The parental DNA isolated from one set’ of gradients was repeatedly recombined and banded on additiona’ sodium iodide gradients to obtain highly enriched parental DNA. Table 3 shows the sequential purification of parental DNA in experiment 3. The purification factors presented in Table 3 represent the degree to which H strands in DNA F were separated from radiolabeled L strands (L*). The purification factor was determined in experiments 3 and 4 from the specific activities and initial ratios of these two components of DNA F and the 3H/32P ratio of the enriched parental DNA. The purification factor for experiment 4 was calculated as follows. The ratio of 3H cts/min: 32P cts/min was 1196 for the purified parental DNA used for la0 analyses. The specific activities of L* and H were TABLE 3 Sequential Centrifuge run number
t Purification $ One tube
puri$cution
of parental DNA in experhent
Parental DNA bg/run)
1
2400
2 3 4
1240 640 -
5 6
130
7 8
-
input
55
factor for fraction 0 (Fig. 5). lost in a centrifuge accident
WCLS
Purification factor
Tubes per run
-
12 6 6 4 3 .>
GO 1120 -
2 2&+
4276 13,zoot
during an attempt
3
to reband.
288
D. R.
STRAYER
AND
I’.
D. BOYER
FIQ. 3. Photograph of a centrifuge tube showing the separation of L and H strands of singlestranded DNA F from expt 4. The H band appears to contain more DNA than the L band because of its proximity to the U.V. light source below the tube. A needle was inserted above the H band to remove the L band. The H bands were combined and rerun in NaI gradients repeatedly until the desired purification was obtained. The H strands of DNA F from all experiments were purified in this manner. The light region at the bottom of the tube was produced by reflected light from the ultraviolet light source below the tube.
1250 cts/min 32P and 1410 cts/min 3H per microgram of DNA, respectively. Therefore, the ratio of H DNA/L* DNA was 1060. The composition of DNA F was 38% H DNA and 62% L DNA (Table 1). Only 723% of L DNA was synthesized during the 32P, and H2180 exposure (Table 2). Therefore, the ratio of H DNA/L* DNA in DNA F was 0.842. The purification factor = 1060/0+342=1260. The parental DNA was not radio-labeled in experiments 1 and 2 to enable study of the replacement of both the thymine and phosphate moieties. The purification of parental DNA in experiments 1 and 2 was not followed sequentially but was determined for the DNA fractionated on the final density gradient. (c) Thymine
and phosphate replacement
in parental
DNA
The final purified parental DNA after banding was separated into fractions and the 3H and 32P content of each fraction determined. Results of such bandings and isotope determinations are given in Figures 4 and 5. The lowest residual 3H and 32P detected in the parental DNA (fractions of greatest purification) from the final sodium iodide gradient and the specific activity of 3H (615 cts/min per pg) and 32P (3750 cts/min per pg) in the L* DNA were used to set
PARENTAL
DNA
INTEGRITY
-c -4 -e - I: - It -2 9 Fractton
number
FIG. 4. Upper limit of thymine and phosphate replacement in each parental DNA strand during one generation of growth (expt 1) : NaI density-gradient fractions of the purified parental DNA were drop collected (40 drops/fraction) through a 24-gauge needle inserted into the bottom of thrk polyallomer tube. This density gradient represents the 6th sequential banding in NaI for this parental DNA. Each fraction was heated at 100°C for 20 min after the addition of 3.0 ml of 0.111NsOH and precipitated at 0°C with trichloroacetic acid and collected: onto Millipore filters. Thv alkali treatment was used to minimize any possible RNA contamination. Each filter was washed 3 times with 10 ml of ice-cold 0.03 M-trichloroacetic acid before drying and measurement of 3H and 32P per fraction. The DNA content of each filter was measured by the diphenylamine reactirtlr as described in Materials and Methods. -e-e--, DNA; ---C-o--, thymine; --G-o---, phosphate.
Frochon
number
FIG. 6. Upper limit of thymine and phosphate replacement in each parental DNA strand during 1 generation of growth in expt 2: NaI density-gradient fractions of banded parental DNA were drop collected (25 drops/fraction) and analyzed as in expt 1 (Fig. 4). This density gradient represents the 7th sequential banding in NaI for this parental DNA. -@-a--, DNA; -m-O--, thymine; -O-O-, phosphate,.
290
D. R.
STRAYER
AND
P. D. BOYER
the upper limits on the thymine (1 per 20,000 thymine residues) and phosphate (1 per 6600 bases) replacement per strand in parental DNA during normal replication in experiment 1 (Fig. 4). Enrichment of parental DNA decreased at lower densities, consistent with an increasing contribution from the L* DNA strand. In experiment 2 (Fig. 5) the specific activity of 3H (110,400 cts/min per pg) and 32P (29,600 cts/min per pg) in L * DNA set the upper replacement limits in each parental DNA strand at 1 per 10,000 thymine residues and 1 per 7000 bases, respectively. The higher upper limits for phosphate replacement compared to thymine replacement in these two experiments may reflect the larger number of cellular components which contain phosphate and could be sources of trace contamination. (d) Ia0 replacement in parental DNA Experiments 3 and 4 were designed to detect possible hydrolytic cleavage of the phosphodiester backbone of parental DNA that might occur more frequently than thymine or phosphate replacement. The cells containing 13C, 15N-labeled DNA were exposed to H, Ia0 for approximately one generation, Hydrolytic cleavage and reformation of the phosphodiester backbone by Ha la0 during this exposure could permit the incorporation of Ia0 into the phosphate moiety of the parental DNA. Detection of an increased level of Ia0 would place limits on the frequency of such hydrolytic cleavage. In experiment 3 DNA F was purified in two separate series of sodium iodide gradients. Table 3 follows the 13,200-fold purification of 7.4 mg of DNA F through eight sequential centrifuge runs. The specific activity of DNA L* and DNA H was 80,400 cts/min 3H per pg and 40.8 cts/min 32P per pg, respectively. Analysis of one tube from the eighth centrifugation is shown in Figure 6. The upper limit for thymine replacement was 1 per 8700 thymine residues. The remainder of DNA F (14 mg)
Frctctkm number FIG. 6. Upper limit of thymine replacement per thymine residue in parental DNA during 1 generation of growth in expt 3: NeI density-gradient fractions of the purified parental DNA were drop collected (30 drops/fraction) and analyzed as in experiments 1 and 2. -O-O--, DNA; - q - q -, thymine.
PARENTAL
DNA
INTEGRITY
291
was purified 620-fold in a second series of sodium iodide gradients with recovery of 720 pg of purified parental DNA. The ratio of 3H cts/min : 3aP cts/min in this purified parental DNA was 4.81. The I*0 content of this purified parental DNA (DNA H) is shown in Table 4. There was no significant difference between the ls0 content of the E. co& carrier DNA and DNA A and B. However, the Ia0 content of the parental DNA was significantly greater than that of the carrier and control DNAs (p <0.02). The parental DNA contained 0.2152 atom y0 180 after correction for carrier dilution. The 3H radioactivity detected in this purified parental DNA set the L* strand contamination at a maximum of 0.24%. The L* DNA was synthesized during the H,180 exposure of the parental DNA in media P which contained 15 atom y0 excess H,180. Since only the branch phosphate oxygens are derived from media phosphates. which rapidly exchange oxygens with media H,O, the L* DNA phosphates contained 0.75 atom O/oexcess I80 (Richards & Boyer, 1966). Therefore, the contribution by L* to the l”b content of the parental DNA was only about 0*0018 atom %. The actual la0 content of the parental DNA after the H,lsO exposure was 0.2134 atom s,, or 0.0105 atom 7; greater than before the exposure. This difference is consistent with one hydrolytic cleavage per 150 bases per strand if all the oxygens which enter the DNA phosphate at the time of cleavage are retained upon resynthesis of the phosphodiester bond. If, however, as seems likely the three phosphoryl group oxygens become equivalent, the entering oxygen has only a 213 chance of remaining in the DNA with reversal of the hydrolytic reaction and closure of the nick in the phosphodiester backbone. In this case, the frequency of hydrolytic cleavage per strand would be I per 100 bases or greater than @I-fold more frequent than the upper limit for thymine replacement. The increased la0 content detected in the parental DNA was not secondary t&oan elevated ls0 content in the [13C]glucose used to density label the parental Dn’A, since the l*O content of the [13C]glucose was not statistically different from the la0 cement of normal abundance glucose. In addition, control DNA grown at’ the same time in the same ]13C]glucose media as the parental DNA, but not exposed to the H,180, had no increase in Ia0 content. TABLE 4
IlNA source
Atom 4’ ,” 180
Average atom SC,
DNA (rg)
Carrier (NT)
DNA B DNA A
275 276
1100 1100
0.2040 0.2017
0.2029
DNA H (parental)
360
1070
0.2066 0.2065
0.2065
-
2300
E. coli carrier DNA
0.2022 0.2046 0.2041
0.2036
Each DNA sample was diluted with E. coli DNA (carrier) to obtain a total of approx. 1400 pg of DNA per sample for IsO analysis. Each figure in the atom Oc / IsO column represents a simultaneous but independent work-up of each DNA sample from the alkaline permanganate oxidation step through mass spectrometrio analysis of its IsO content. DNA B was extracted from cells removed at (c) Fig. 1 (expt 3) just before the Hz I80 exposure. DNA A was extracted from E. coli lST- cells grown in 11 of medium P. DNA H was the enriched parent)al DNA.
292
D.
R.
STRAYER
AND
P.
D.
BOYER
Contamination of the purified parental DNA by non-DNA phosphate compounds containing ls0 was unlikely because of the purification procedures used. For example, these procedures should separate DNA from any RNA, particularly since any RNA present would be hydrolyzed by treatment with 0.1 M-NaCH at 100°C for 20 minutes before trichloroacetic acid precipitation of the DNA onto Millipore filters. Nonetheless, a more positive control on possible 180-phosphate contamination was felt necessary. Experiment 4 was performed under conditions that allowed critical evaluation of possible contamination of the parental DNA by phosphates containing 180, since the H,180 exposure of the parental DNA was done in the presence of 32P,. The parental DNA (14 mg) was purified 1260-fold away from 32P-containing materials with a recovery of 1.31 mg of purified parental DNA (see section (b), above, for calculation of purification factor). Medium P contained 4.6 atom o/o excess H2180. The ratio of 3H cts/min: 32P cts/min in the purified parental DNA was 1196. The atom o/o ls0 contents of DNAs in experiments 3 (Table 4) and 4 (Table 5) are not directly comparable, since (1) experiments 3 and 4 were significantly different in design, (2) the H2180 content of medium P was threefold higher in experiment 4 and (3) different carrier DNAs were used in each experiment. The 180 content of parental DNA from experiment 4 is seen in Table 5. The 180 content of the non-isotopic control DNA (DNA A) was not significantly different from the calf thymus carrier DNA. The 180 content of the parental DNA was significantly greater than DNA A (p < 0.005). The 180 content of the parental DNA after correction for carrier dilution and L* strand contamination was 0.2077 atom y. or 0.0052 atom y. greater than DNA A. Assuming three equivalent phosphoryl group oxygens, this difference is consistent with one phosphodiester bond cleavage per 520 bases per round of chromosome replication in each parental DNA strand. TABLE 5 180 content of purified parental DNA in experiment 4 DNA
DNA
SOUITX
DNA
(P?4
Carrier hi9
Atom y0 160
A
215
960
DNA H (parental)
440
720
0.2060 0.2052 0.2056
0.2052
-
1200
0.2027 0.2022 0.2027
0.2025
Thymus carrier DNA
0.2026 0.2017
Average atom o/0 0.2022
Each DNA sample was diluted with calf thymus DNA (carrier) to obtain a total of approx. 1200 pg of DNA per sample for I80 analysis. Each figure in the atom o/o IsO column was determined independently as in Table 4. DNA A was extracted from cells removed at (a) Fig. 1 (expt 4). DNA H was the enriched parental DNA.
4. Discussion These experiments place upper limits on the incorporation of thymine (1 per 10,000 to 20,000 thymine residues) and phosphate (1 per 7000 bases) into each parental E. coli DNA strand during normal replication. The upper limit found for
PARENTAL
DNA
INTEGRITY
293
thymine replacement was lower than that for phosphate replacement. The incorporation rate found for thymine into parental DNA is less than or equal to 50 to 100 thymine residues replaced per DNA strand in the E. coli genome. The rate of incorporation of the three other bases into parental DNA was not studied. The discrepancy between the thymine and phosphate replacement could possibly reflect an increased frequency of incorporation of one or more of the other bases compared to thymine. A more likely explanation is the failure to purify the parental DNA away from nonthymine containing phosphate compounds. These limits reached for phosphate and thymine replacement are, as far as we are aware? the lowest yet observed, approaching one to three per Worcel loop. The increased level of 180 detected in the phosphodiester branch oxygens of parental DNA after replication in media containing H,O enriched in 180 is consistent with hydrolytic cleavage of the phosphodiester bonds (1 per 100 to 500 bases per parental strand). The increased parental DNA l*O content cannot be explained by endonuclease activity during the DNA isolation procedure prior to separation from H,laO. Denaturation of such frequently nicked parental DNA would yield low molecular weight fragments incompatible with the DNA bands obtained (Fig. 3) and t’he enrichment of parental DNA achieved on sodium iodide gradients. Therefore, the majority of the phosphodiester bond cleavages detected must have been resealed prior to DNA isolation. Although the molecular mechanism of the cleavage and resynthesis of the phosphodiester bonds is unknown, a DNA relaxation protein like w (Wang, 1971) could play a possible role in DNA replication. w is a protein found in E. coli that acts like a .‘swivel” removing negative superhelical turns from closed circular DNA in a stepwise manner. Unwinding of DNA during replication produces positive superhelical turns. The failure of Wang to detect relaxation of positive superhelical turns in but does not eliminate w as a bacteriophage lambda DNA by w is bothersome, possible swivel for DNA replication in E. coli. DNA relaxation proteins have also been found in mouse cells (Champoux & Dulbecco, 1972; Vosberg et al., 1974). in eggs of Drosophilia melanogaster (Baase & Wang, 1974) and in human tissue culture cells (Vosberg et al., 1974; Keller, 1975). The unwinding is probably accomplished by a rapid nicking and resealing mechanism that appears to proceed without a’ requirement for external energy (Wang, 1971). Wang has proposed a covalent81y linked protein-DNA intermediate which stores the phosphodiester bond energy during relaxation. The reaction is reversible and the phosphodiester bond is restored as w leaves. Such a mechanism would not be expected to give an increased la0 content in parental DNA. DNA gyrase, an enzymatic activity isolated from E. co&, introduces negative superhelical turns into covalently closed circular DNA in an ATP-dependent reaction (Gellert, Mizuuchi et al., 1976) and appears to be essential for the replication of circular DNA in E. coli (Gellert, O’Deaet al., 1976). Recentevidencesuggests that DNA gyrase consists of two components : (1) a nalidixic acid-sensitive nicking/closing activity (ATP independent), and (2) a coumermycin-sensitive supercoiling activity (BTP dependent) (Sugino et al., 1977 ; Gellert et al., 1977). The nicking/closing activity is distinct from u protein and can relax both negatively and positively supercoiled DNA. DNA gyrase, like w protein, may utilize a covalently linked protein-DNA intermediate that is competent for resealing (Sugino et al., 1977 ; Gellert et al., 1977). However. the cxist’ence of such intermediates for DNA gyrase and w prot,ein remains
294
D.
R. STRAYER
AND
P. D. BOYER
tentative. It is possible that energy is stored by an enzyme molecule which has been “activated” by the cleavage reaction to a higher energy state (Wang, 1971). Conformational or structural features in the enzyme protein could favor resynthesis of the phosphodiester bond. For example, the formation of an acyl phosphate from P, without phosphorolysis of a pre-existing covalent bond has been reported by this laboratory (Dahms et al., 1973). The formation of a non-covalent “activated” DNA gyrase or w protein-DNA intermediate could result in the increased ls0 content we observed in parental DNA. A second mechanism which could explain the increased la0 content of parental DNA would utilize an endonuclease and ligase activity (Sebring et al., 1971). DNA replication would proceed with formation of positive superhelical turns until the parental strands could not unwind any further. At this point replication would temporarily cease. An endonuclease, possibly recognizing a strained region of the DNA molecule, could introduce a hydrolytic cleavage that would allow relaxation of the strain and replication could proceed. An alternative possibility would be that endonucleolytic cleavage and ligase repair occur frequently along the length of the DNA molecule and thus prevent conformational strain and cessation of DNA replication. This model would predict an accumulation of single-strand cleavages in DNA with cellular ATP depletion consistent with the results reported by Hilton t Walker (1977). Single-strand breaks could also facilitate the local separation of DNA strands, which occurs during selective gene transcription (Frenster, 1976). Thus, it is possible that events accompanying transcription (Riva et al., 1970) or other metabolic processes could be responsible for some or most of the la0 incorporation we have observed. The authors are very grateful to Dr Ragini Anet for many helpful discussions and for her fine experimental efforts that helped in initiation of these experiments many years ago. Thanks are also due to MS Kerstin Stempel for careful lsO analysis. This work was supported in part by contract E(O4-3)-34) of the Atomic Energy Commission, U.S. Energy Research and Development Administration.
REFERENCES Anot, R. & Strayer, D. R. (1969). Biochem. Biophys. Re.s. Commurr. 37, 52-58. Baase, W. A. & Wang, J. C. (1974). Biochemistry, 13, 4299-4303. Burton, K. (1956). Biochem. J. 62, 315-323. Champoux, J. J. & Dulbecco, R. (1972). Proc. Nat. Acad. Sd., U.S.A. 69, 143-146. Dahms, A. S., Kanazawa, T. & Boyer, P. D. (1973). J. Biol. Chem. 248, 6592-6595. Delius, H. & Worcel, A. (1974). J. Mol. Biol. 82, 107-109. Dische, Z. (1930). Mikrochemie, 8, 4-32. Frenster, J. H. (1976). Cancer Res. 36, 3394-3398. Gellert, M., O’Dea. M. H., Itoh, T. & Tomizawa, J. (l!J76). Proc. iliat. Acad. Xci., U.S.A. 73, 4474-4478. Gellert, M., Mizuuchi, K., O’Dea, M. H., ltoh, T. & Tomizawa, J. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 4772-4776. Gellert, M., Mizuuchi, K., O’Dea, M. H. & Nash, H. A. (1976). I’roc. Nat. Acad. Sci., U.S.A. 73, 3872-3876. Hilton, J. & Walker, M. D. (1977). Biochem. Biophys. Res. Commun.. 77, 9099914. Keller, W. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 2550-2554. Marmur, J. (1961). J. Mol. Biol. 3, 208-218. Messelson, M., Stahl, F. W. & Vinograd, ,J. (1957). Proc. Nat. Acad. Xci., U.S.A. 43, 581-588. Richards, 0. C. & Boyer, P. D. (1965). J. ,Vol. Biol. 11, 327-340. Richards, 0. C. & Boyer, P. D. (1966). J. MoZ. BioZ. 19, 1099119.
PARENTAL
DNA
INTEGRITY
“!I5
Kiva, S., Cascino, A. & Gciduschek, E. P. (1970). .J. Mo7. J3io7. 54, IO3 I I!). Sebring, E. D., Kelly, T. tJ., Thoren, M. M. CycSalzman~ X. I’. (1971). .I. lyirol. 8, 4i8 490. Stoningt~on, 0. C. C%P&ijohn, D. E. (1951). I’roc. Nat. ilcntl. ,C&, 1,T.S.A. 68, 6-9. Srlgino, -4., Peeblcs, (:. L., Krewrr, K. N. & C!oxzarelli, N. Ii. (1977). Proc~. n’at. rlrad. Sci., U.S.A. 74, 4767-4771. Tornizawa. J. & Ogawa, T. (1968). C’old 8prin.g Harbor ~Symp. Quant. Bid. 33, 533. 551. Vosbrrg, H. I’.. Grossman, L. I. & Vinograd. .J. (1974). Ferl. J’roc. Fed. a+imv-. SW. ET/J. Rid. 33, 1356. Weang, ,J. C!. (I 971). .I. Mol. Bid. 55, 523-533. IVorcel, A. & Burpi, E. (1972). J. Mol. Bid. 71, 1277147. \~~orcr:l. A. & Hlugi,
E. (1974). .I. Mol.
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82, 91- 105.