Destabilized secondary structure of newly replicated HeLa DNA

J. Mol. B&Z. (1970) 49, 157-170

Destabilized Secondary Structure of Newly Replicated HeLa DNA JOEL I?. HABENER~, BARBARA S. BYNUM AND JOSEPH SHACK: Laboratory of Physiolugy, National Cancer Institute National Institutes of Health, Bethesda, Md. 20014, U.S.A. (Received 28 July 1969, and in revised form 11 December 1969) DNA, extracted by a variety of methods from intact HeLa cells pulsed with [3H]thymidine and also from the washed nuclei and dispersed nucleoprotein derived from them, was examined for the presence of single- and double-stranded forms by hydroxyapatite chromatography, zonal electrophoresis and susceptibility to exonuclease I. A substantial portion of the newly replicated DNA can be extracted from intact cells or nuclei in a single-stranded form by certain of the procedures, while DNA extracted from the dispersed nucleoprotein by the same procedures is entirely double-stranded. DNA extracted from whole cells by various other procedures is also almost entirely double-stranded. These results suggest that a portion of the newly replicated DNA is present in the nucleus not as free single strands but in a “destabiliied” state and that, depending on the conditions of extraction, it can be converted to either the single- or double-stranded form. The results further suggest the possibility that factors operative in the intact nucleus but not in the nuclear lysate maintain the newly replicated DNA in this destabilized state.

1. Introduction Information about the physical state of replicating DNA would contribute to a more complete understanding of the replication process. It is presently known that mammalian DNA is replicated semi-conservatively (Simon, 1961) in discontinuous segments or replicons (Painter, Jermany & Rasmussen, 1966; Taylor, 1968) which appear to grow in divergent directions from common initiation points on the chromosome (Huberman & Riggs, 1968). Autoradiographs (Cairns, 1966; Huberman & Riggs, 1968) have shown fork-like structures at the replicating site, supporting the theory that the replicating template strands unwind, separate and form new double-stranded molecules through base pairing with complimentary nucleotides. However, the mechanisms operating to unwind the template double-helix and to assemble and rewind the newly synthesized helix are not understood. One approach to obtaining information about mechanisms of DNA replication is to examine the secondary structure of newly replicated DNA: a loosening or partial separation of the strands might be predicted from the above-mentioned considerations. Indeed, a number of reports indicate that part of the newly synthesized DNA extracted from bacteria is “partially denatured” (Kidson, 1966) or single stranded (Okazaki, Okazaki, Sakabe, Sugimoto & Sugino, 1968; Oishi, 1968). It should, however, be noted t Present address: Endocrine Unit, Massachusetts General Hospital, Boston, Mass. 02114,U.S.A. $ To whom requests for reprints should be sent. 167

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J. F. HABENER,

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AND

J. SHACK

that Yudelevich, Ginsberg & Hurwitz (1968) report the isolation of newly formed bacterial DNA in double-stranded form. Results with mammalian cells have &o been varis,ble. Paoletti, Dutheillet-Lamonthbie, Jeanteur $ Obrenovitch (1967) have suggested, on the basis of its affinity for methyl&d albumin, that & portion of newly synthesized DNA isolated from ascites cells has less secondsry structure than the pre-existing DNA. However, from partition studies on hydroxyapatite, Tsukada, Moriyama, Lynch & Lieberman (1968) conclude that the newly replicated DNA extracted from rat liver nuclei is entirely double-stranded while Painter & Schaefer (1969) fmd that a portion of the newly formed DNA of HeLa cells is single-stranded. Our own work, which is reported here, appears to resolve some of these discrepancies. We have extra&d DNA from pulse-labeled HeLa cells by TVvariety of methods over a range of temperatures and present evidence indic&ng that a substturtial portion of the newly replicated DNA may be isolated in either single- or double-stranded form depending upon the conditions of extraction. The results suggest that this particular fraction exists within the cell in what may be called a transient destabilized state. A preliminctry account of the present work has already been presented (Habener, Bynum & Shack, 1969).

2. Materials and Methods (a) Radioactive labeling of HeLa ceUa A suspension culture of HeLa S3 cells (Puck, Marcus & Cieciura, 1966) WBBkept in log phase at 37°C with Eagle’s MEM medium mod&d for spinner culture (Eagle, 1969) and supplemented with 5% home serum. Penicillin (100 units/ml.) and streptomycin (100 &nl.) were also added to the medium. Pre-exLting DNA was labeled by growing the cells for a generation time in [14C]thymidine (New England Nuclear Corporation, Boston, Mass., 10 ma/m-mole, 0.025 pa/ml.). Cells were centrifuged at 37°C reauspendecl in fresh medium at a concentration of from 1 to 3 x lo* cells/ml. and allowed to inoubate at 37% for 1 hr before pulse-labeling for OS to 3 min with [3H]thymidine (New England Nuclear Corporation, 16.2 to 18.2 c/m-mole, 2 &ml.). Incorporation of [aH]thymidine W&B stopped after the specified time either by pouring the cells into a 0-S vol. of frozen medium or by lysiug the cells in 1% sodium lauryl sulfate. Samples of the pulse-labeled cells were chased by adding a SO-fold exceed of unlabeled thymidine directly to the cell suspension containing the [3H]thymidine; this W&Bsu&ient to reduce the rate of [3H]thymidine incorporation by SO- to loo-fold. (b) Prepamtim of nuclei and d&per.& nucikoprotein About lo8 cells were washed twice by centrifugation in 100 to 200 ml. of phosphate buffered saline (036% NaCl, pH 7.6) at 3°C. The cell pellet was then resuspended in OS the wash volume of phosphate-buffered saline and the cells were lysed by adding an equal volume of 1% (v/v) Non-Idet P40 detergent (Shell Chemical Co., New York, N.Y.) in 0.076 M-NaCl-0.024 M-EDTA (pH 8.0) and allowing the mixture to stand at 0°C. Cell lysis was complete within 15 to 20 min. The nuclei were washed twice in 100 ml. of a solution containing equal volumes of phosphate-buffered saline and 0.075 ~-N&1-0*024 M-EDTA (pH 7.6) at 3°C. Nucleoprotein was prepared by dislodging the nuclear pellet with a 10 ml. stream of 0.25 mu-EDTA (pH 8.2) and pouring immediately into a Waring blender containing 200 to 300 ml. of 0.26 maa-EDTA at 0 to 2’C; the blender was run at 9000 rev./min for 20 sec. After this procedure more than 90% of the [“Cl- and C3HjDNA radioactivity and of the protein aa determined calorimetrically (Lowry, Rosebrough, Farr & Randall, 1961) remained in the supernatant after centrifuging at 20,000 g for 30 min. (0) Eztraotion of DNA Three different methods were used to extract DNA. In each case, temperature of extra&ion refers to that temperature at which stirring with sodium para-amino salicylate was carried out.

DESTABILIZED

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(i) Method I. Mod@& Kirby procedure I (Kirby, 1964) Cells were lysed by pouring a sample of the cell suspension (at 37°C) containing 1 to 2 x 1Oe cells in 26 to 60 ml. of complete medium into an equal volume of 2% sodium lauryl (flask immersed in a water bath at temperature of extraction) sulfate-O.004 M-EDTA while stirring with a Teflon propeller; after 1 min, enough sodium par&-amino salicylate was added to make a 6% solution, and stirring was continued for 30 min at the desired temperature. In a slight variation of this procedure, it was found that results were essentially the same if sodium para-amino salicylate was added to the detergent solution before adding an equal volume of the cell suspension. (ii) Method II. Modi$ed Kirby procedure II The cells, after rapid chilling to O”C, were centrifuged into a pellet and resuspended in 8 to 10 vol. of water (at about 24%) in order to swell the cells. An equal volume of warmed 12% sodium para-amino salicylatte (previously brought to the temperature of extraction) wa8 added snd the cells were disrupted in a Potter-Elvehjem homogenizer for 6 min at temperatures ranging from 0 to 80°C. (iii) Method III.

Modijkd Berm-Thomm procedure (Berm & Thomas, 1965) Chilled cells were centrifuged, washed, resuspended in 20 to 30 vol. of 1 x SSC (0.15 MN&1-0*016 r+sodium citrate), O-002 M-EDTA and lysed by stirring gently in 1% sodium lauryl sulfate at room temperature. The lysate was incubated at 37°C for 16 hr with pronase (2 mg/ml., Calbiochem, Los Angeles, Calif.), enough sodium para-amino salicylate was added to make a 6% solution and the mixture was stirred for 30 min at the desired temperature. The lysates prepared by each of the above 3 methods were then extracted for 40 min at 26°C with a solution consisting of 88% phenol, 12% nt-cresol and 0.1% 8-hydroxyquinoline. The extracts were chilled, centrifuged, and the upper aqueous layer was exhaustively dialyzed against 0.01 M-phosphate buffer (pH 6.8) in preparation for hydroxyapatite chromatography. In several preparations 0.2 to 0.6 mg of heat-denatured carrier calf thymus or unlabeled HeLa DNA was added either to the cell sample before lysis, or to the lysate before phenol extraction. The results were unchanged by the addition of denatured carrier DNA. (d) Hydroxyapatite

chromatography DNA in 0.01 M-phosphate buffer was sheared by sonication and chromatographed on columns according to the method described by Bernardi (1965). Hydroxyhydroxyapatite apatite was prepared by the method of Miyazawa & Thomas (1965) or was purchased from Bio-Rad Laboratories, Richmond, Calif. Approximately 100 pg of DNA was eluted from 1.0 to 1.6 ml. packed volume of hydroxyapatite with a linear phosphate buffer gradient. Elutions were usually done at 25°C but in some cases elution was carried out at a higher temperature throughuse of a thermostatically controlled water jacket. 1.5 ml. fractions were collected at a flow rate of about 0.6 ml./min. Before hydroxyapatite chromatography, the DNA solutions were cooled to 20°C and sonicated for 20 set in a Branson LS76 SonZier (Branson Instruments, Inc., Stamford, Corm.) at a power setting of 4. Only slight heating occurred under these conditions. Before sonication the size of the DNA ranged from about 106 to lo* daltons and flow rates were very slow and recovery poor. Sonication reduced the size to about 0.6 to 1.0 x IO6 daltons and resulted in even flow rates and almost complete recovery of radioactivity from the hydroxyapatite columns.

DNA in the fractions of added carrier (O*l”h cold 6% trichloroacetic for 20 min. Particularly soluble 3H radioactivity further examination of

(e) Radioactivity determinationa was precipitated with cold 6% trichloroacetic acid in the presence calf thymus DNA and l*6o/o bovine serum albumin), washed with acid and then extracted with hot 5% trichloroacetic acid at 90°C with short pulses a significant amount of trichloroacetic acidwas eluted from hydroxyapatite just before denatured DNA; this fraction was carried out as described later. Appropriate samples

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(approx. O-1 ml.) of each fraction were counted in 10 ml. of scintillation fluid (00% toluene, 40% ethylene glycol monomethyl ether, 8.0% naphthalene, 0.4% PPO, O-006% POPOP) in a Packard TriCarb Liquid Scintillation Spectrometer (Packard Instruments Co., Inc., LaGrange, Ill.). Counting data were recorded on punch tape and efficiency corrections, sH/14C ratios and percentage of total radioactivity were determined with the IBM 1020 computer (International Business Machines Corporation, White Plains, N.Y.). Plots were generated by a Dymec digital data translator and a model 2D2 X-Y recorder (F. L. Mosley Co., Pesadena, Calif.). (f) Treatment with exonucbe I Eacherich& co.& exonuclease I (Worthington Biochemical Corp., Freehold, N.J.) was used to digest certain DNA prepamtions and hydroxyapatite fractions according to the procedure described by Lehman & Nussbaum (1964). The incubation mixture volume was 376 ~1. containing O-06 M-glycine buffer (pH g-56), 0.002 aa-2-mercaptoethanol, 0.006 ar-MgCl,, 2-5 units of exonuclease I and 6 to 100 pg of DNA. The reaction was initiated by addition of enzyme and the incubtttion was carried out at 37°C for 00 min. At 30 min the reaction was about 80% completed compared to the final value at 00 min. At the end of the incubation period 100 to 200 4. was removed and treated in the presence of added carrier calf thymus DNA with cold 6% trichloroacetic acid. Acid-soluble radioactivity was then measured in the trichloroacetic acid supernatant and expressed as the percentage of total acid precipitable radioactivity present at zero time. (g) Zonal electrophoresis Electrophoretic separations of unsonicated DNA were carried out in an LKB model 3340 column electrophoresis apparatus (LKB-Produkter AB, Stockholm) as previously described (Shack & Bynum, 1964). Descending electrophoresis was used at 3°C in a stabilizing gradient of 2 to 42% sucrose and the buffer was 0.002 M-sodium phosphate, pH 7.00 f 0.02. Following electrophoresis, fractions were collected and precipitated with trichloroacetic acid before counting radioactivity. (h) Bio-Gel jiltrakiun and Dowex-1 chromatography The frclotions from hydroxyapatite which were found to be trichloroacetic acid soluble were examined by filtration on Bio-Gel P2 (Bio-Rad Laboratories) in 0.01 aa-‘Iris buffer (pH 7.5) at 25°C and by chromatography on Dowex-I (Bio-Rad AG l-X8, Bio-Rad Laboratories) according to the method of Grav t Smellie (1963). Fractions (l-95 ml. each) were collected and examined for ultraviolet absorption and radioactivity. (i) Treatment with a&dine phoaphutaae Fractions from Dowex-1 columns or from hydroxyapatite chromatography were pooled, desalted, ooncentrrtted, and adjusted to pH 7-S in 0.5 aa-ammonium bicarbonate buffer. E. wli alkaline phosphatase (Worthington Biochemical Corp.) was then added (O-1 unit/ ml.) and the mixture was incubated at room temperature for 1 hr.

3. Results (a) Separation

of single and double strands of newly repkated DNA on hydroxyapatite

The separation of DNA extraoted from pulse-labeled cells into single (I) end double (II) strands by hydroxyapatite chromatography is shown in Figure 1. The extractions in this case were done at several different temperatures using method I. The amount of 3H-labelod newly replicated DNA appearing as single strands increases with the temperature of extraction (32% at 80%) while the bulk of the DNA labeled with 14C remains almost entirely in the double-stranded form (less than 5% of single strands at 80°C) until the thermal denaturation temperature is reached at 90°C. The fact that at 90°C both newly replicated DNA and old DNA are equally distributed between the

DESTABILIZED

NEWLY

REPLICATED

HELA

DNA

161

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3.2

0.1 -2 0

;

% 0.3 B

@2

0.1

0

4

8

12

16

20

24 0

4

8

I2

16

20

24

0

Fraction no. FIG. 1. Hydroxyapatite chromatography of DNA extracted from HeLs cells pulse-labeled for 3 min with C3H]thymidine. DNA was extracted by method I at (a) 1OT; (b) 2577; (c) 80%; (d) 90°C. Approximately 100 pg was eluded from 1.5 ml. packed volume of hydroxyapatite at 25’C and assayed for radioactivity aa described under Materials and Methods. Rtldioactivity counted: 3H, 1563 to 2613 cts/min; I%, 4381 to 9211 cts/min. (-•--a-) 3H radioactivity; (-O-O-) l*C radioactivity; (----) phosphate buffer concentration; I, single stranda; II, double strands.

single- and double-stranded fractions suggests that a portion of the newly replicated DNA may actually be more resistant to heat denaturation than the bulk of the DNA. Shorter periods of pulse labeling (0.5 min) resulted in a larger fraction (40%) of singlestranded DNA. Figure 2 shows a progressive decrease in the amount of the singlestranded fraction during incubation with an excess of non-radioactive thymidine, thus establishing that the single-stranded fraction is a property of the newly replicated state. Evidence that single-stranded DNA was not created by the sonication procedure was obtained by zonal electrophoresis of unsonicated DNA; in every case separation into characteristic native and denatured components was essentially identical to those seen on hydroxyapatite. A typical experiment is shown in Figure 3. Further confirmation of the secondary structure of the hydroxyapatite fractions was obtained by 11

182

J. F. HABENER,

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r

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6

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12

14

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22

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Froctton no. FIG. 2. Hydroxyapatite chromatography of pulsed-chased HeLa DNA. A suspension of cells at a concentration of 2.7 x IO6 cells/ml. waspulse-labeled with [3H]thymidine arg described in the text. At 3 min 8 50-fold excess of unlabeled thymidina was added. Samples of the suspension culture were with&awn at the times indicated and DNA ~88 extracted at 80°C by method: I. (-O-O-) 0.5min; (-@-a---) 3min; (-A-A-) Emin; (-A---A-) 3Omin; (-•-~--) 6Omin; (----) phosphate buffer concentration. Tot81 radioactivity counted: 2 to 4 x IO9 cts/min.

with E. coli exonuclease I, which is known to degrade only single-stranded DNA (Lehman & Nussbaum, 1964) (shown in Table 1). The percentage of single strands estimated from enzyme treatment is in every case in good agreement with values obtained from fractionation of either unsonicated or son&ted DNA, a further indication that sonic&ion does not significantly increase the extent of denaturation. treatment

(b) Dependence of newly replicated single strands upon the me&d anoTtemperature of extraction The yield of single strands depends on both method and temperature of extraction, For example, extraction by methods II end III yields only relatively small amounts of single-stranded [3H]DNA at 25°C (Fig. 4); by contrast, nearly 20% of the [3H]DNA extracted by method I at the B&metemperature is single stranded (cf. Fig. 1). Figure 6 shows how the fraction of newly replicated DNA eluted from hydroxyepatite aa single strands depends on temperature and method of extraction. The ordinate here, as well as in Figure 7, gives the percentage of single-stranded [3H]DNA minus the percentage of single-stranded [14C]DNA. The values for single-stranded [14C]DNA were for methods I and II less than 2% below 70°C and less than 5% at 80°C. With method III

no.?

III II II II I I I I

Isolation method

0 60 60 70 70 80 80 80

of

6.8$ O-6$ 1.11 2*6$ 3.08 4.6$ 5.65 5.2$

1°C

by a 60-min chase.

8.8 9.3 2.0 18.4 34 29.7 26.2 6.0

3H

Radioactivity in single-stranded fraction (%I

acid-soluble

3H

94 96 100 99 100 -

“C

5.6 0.2 0.9 3.6 6.3 6-O 7.9 11.8 O-8 22.6 32 7.7

68 77 62 71 66 -

“C

Single-stranded fraction

trichloroacetic

I

3H

whole preparation

‘$/JRendered

of pulse-labeled DNA to E. coli exonhe

t P indicates a 3-min pulse, PC a 3.min pulse followed 1 Fractionated on hydroxyapatite. 8 Fractionated by zonal electrophoresis.

14-P 15-P IS-PC 16A-P 16B-P 17A-P 17A-P 17A-PC

Preparation

Temperature extraction (“C)

hweptibility

TABLE 1

2.5 6.8 15.1 8.4 18.2 -

3H

0.3 3.6 3.1 3.8 2.0 -

1%

Double-stranded fraction

by exonucleaae

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164

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50

60

70

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80

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PO

100

Cathode -+ Fraction

no,

FIG. 3. Zonal electrophoresie of DNA pulse-l&&d for 3 min with [aH]thymidine. Approximately 160 pg of DNA, extracted at 8O’T by method I, was electrophoresced (not sonioeted) for 14 hr at 3°C. 2 ml.-fractiona were collected. (--a-*-) aH radioactivity; (-O-O-) ‘“C radioactivity.

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, I6

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4

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no.

8

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I6

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(b)

FIG. 4. Hydroxyapatite chrometogrephy of DNA extracted by methoda II and III from 3 min pulse-labeled HeLa o&s. DNA was extracted from whole cells st 26°C by (a) method II and (b) method III described in the text. Conditions of chromatogrephy and key to symbols are the ~eme aa those described in the legend to Fig. 1.

the value for single-stranded [14C]DNA varied between 3 and 7% for different preparations with no dependence on temperature; values for ([3H]DNA minus [14C]DNA) were in the range 2 to 5%. The data shown in Figure 5 suggest that the newly replicated DNA may exist in the cell not as free single strands but in a destabilized state and that, depending onextraction conditions, it can be converted into either the single-stranded or double-stranded form during isolation.

DESTABILIZED

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165

DNA

01 0

IO

20

30

40

50

Temperature

60

70

8

(“Cl

FIQ. 5. Dependence of yield of newly replicated single-stranded DNA on method and temperature of extraction. HeLa cells were pulse-labeled for 3 min and DNA, extracted by the specified method, was separated into single (denatured) and double strands on hydroxyapatite. DNA was prepared by: (-@-a-) method I; (-O-O-) method II; (-u-m-) method III.

(c) Dependence of newly re$icated single strands upon fraction of cell from which DNA is extracted In order to ascertain whether the secondary structure of the newly replicated DNA might depend upon a property of the intact cell or nucleus, washed nuclei and dispersed nucleoprotein derived from them were also extracted by method I and studied on hydroxyapatite. Results obtained by extraction at 80°C are shown in Figure 6. The

24 0

(0)

Frcction

4 no.

8

12

16

20

24

(b)

FIG. 6. Hydroxyapatite chromatography of DNA extracted from nucleonrotein. HeLa cells were nuke-labeled for 3 min and DNA was I. For key to symbols see the legend to Fig. 1. (a) Washed nuclei; sH sH 14C radioactivity (10,091 cts/min); (b) dispersed nuoleoprotein; 1% radioactivity (12,733 cts/min).

washed nuclei and dispersed extracted at 80°C by method radioactivity (2445-cts/min), radioactivity (3529 cts/min),

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J. F. HABENER,

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J. SHACK

dependence of the amount of single-stranded DNA on extraction temperature is shown in Figure 7. Results for DNA extracted from washed nuclei are close to those obtained with DNA extracted from whole cells. However, extraction of the dispersed nucleoprotein preparation yielded only a double-stranded fraction. Since the nucleoprotein preparation is simply a mechanical lysate of the nuclei it contains all of the constituents present in the intact nucleus; the only change in the system other than dilution has been a disruption of the nuclear structure. As discussed later, these data suggest the possibility that some aspect of nuclear structure may be involved in maintaining the newly replicated DNA in the destabilized or incompletely hydrogenbonded state.

Temperature

(“C 1

FIQ. 7. Extrtlction of DNA from nuclei and nucleoprotein at different temperatures. DNA w8a extracted by method I, and the amount of eingle-stranded DNA wa 888~yed by hydroxyepetite chromatography. Pulse labeling time w8a 3 min. (0) Washed nuclei; (-m-w-) dispensed nucleoprotein; (0) whole cells (d8t8 from Fig. 6 shown for comperison).

(d) Possibility of loas of newly replicated single sham% We considered the possibility that failure to find a single-stranded fraction when DNA was prepared from cells or nuclei by method II below 40°C by method III, or through the nucleoprotein step might be due to preferential losses of single strands during preparation. This possibility was eliminated by analysis of the ratios of 3H to 14C in the various fractions. Typical data are given in Table 2. It is evident that 3H present in the single-stranded form derived from cells or nuclei appears in the doublestranded fraction obtained from nucleoprotein. We have also carried out several reconstruction experiments in which labeled single-stranded DNA was added at various stages of the extractions, and have subsequently isolated it from hydroxyapatite without preferential losses. (e) Possibility of ~~~rtiizJalteration8 in the seconrkcry strmcture of the newly replicated double-stranded DNA In all of our experiments the newly replicated DNA was well resolved into what appeared to be fully denatured and fully native forms with no evidence of partially

DESTABILIZED

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167

TABLET Comparison of the isotopic ratios of DNA extracted from cel&s,nuclei and nucleoprotein

3H in einglestranded fraction (%)

Preparation

3H/14C ratio of double-stranded fraction

3H/14C ratio of double-stranded fraction calculated by addition of single-stranded 3H fraction

Experiment 1 Whole cells Nucleoprotein

21.2 1.5

160 1.93

1.94 1.96

Experimeni 2 Nuclei Nucleoprotein

20.0 0.9

1.15 1.37

1.39 1.38

Method

I was used here.

denatured intermediates. To determine whether loosening of the secondary structure might exist in newly replicated DNA, once isolated in the double-stranded form by method III, by method II below 25”C, or through nucleoprotein, attempts were made to denature preferentially the newly-formed component by exposure to temperatures sufficient to cause up to 30% denaturation. In every case equal fractions of the newly replicated and previously existing DNA were denatured. Figure 8 shows typical results obtained when DNA extracted from nucleoprotein at 80°C was heated to 60°C at low ionic strength (approximately 12% clenaturation); the absence of preferen-

r 0.3

52 0.2 $ :: 2a 0.1

0

4

8

I2 Fraction

FIG

8. High

temperature

16

20

24

0

no.

hydroxyepatite chromatography of DNA extracted from nucleofrom nucleoprotein at 80°C by method I shown in Fig. 6(b) was chromatogmphed on hydroxyapatite at 60°C. The conditions of chromatography are otherwise &B (2105 cts/min); (-O--O--) described in the legend to Fig. 1. (-O-O---) 3H radi oactivity r*C radioactivity (7162 cts/min); (----) phosphate buffer concentration.

protein. The DNA extracted

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J. F. HABENER,

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tial denaturation indicates that the hydrogen bonding involving the newly-formed segments is quite normal. The good correspondence between amount of singlestranded component estimated from fractionation and exonuolease susceptibility (Table 1) as well as the resistance of the double-stranded component to exonuclease indicate that very few, if any, of these newly formed segments are present as singlestranded ends attached to normal double-stranded material. The partial susceptibility of certain of the double-stranded fractions (cf. 16B-P and 17A-P) is probably due to incomplete resolution of the bands. (f) Tentative identi$c&on of a trichloroacetic fraction from hydroxyapatite

acid-soluble

As mentioned earlier, it was found that a substantial amount of trichloroacetic acidsoluble SH radioactivity was not removed by exhaustive dialysis, adhered to the hydroxyapatite, and eluted just before the single-stranded fraction coincident with a marker 14C-labeled thymidine triphosphate (Fig. 9). Hydroxyapatite fractions 9 to 11 (Fig. 9) pooled and filtered on Biogel P-2 to remove the trichloroacetic acid-precipitable DNA were further analyzed by chromatography on Dowex-I columns (Grav & Smellie, 1963) both before and after treatment with alkaline phosphatase, a monophosphatase speoific for removal of terminal 5’-phosphates. Over 80% of the radioactivity eluted as thymidine triphosphate and was converted by the alkaline phosphatase to material eluting with thymidine thus indicating that the trichloroacetic acid-soluble material contained no internuoleotide phosphates, is not a small polynucleotide, and is most probably thymidine triphosphate. Cleaver & Holford (1965)

TMP

TDP

TIP

6

12 Fraction

16

20

no.

FIG. 9. Hydroxynpatite chromatography of DNA extracted at 70°C from HeLa cells pulsed for 0-b min with [SH]thymidine. Samples of fractions were assayed for radioactivity before (crosshatched area) and after treatment with trichloroacetic acid. The arrow8 show the characteristic elution positione of W-labeled thymidine nucleoeide phosphates which were co-chromatographed with the pulse-labeled DNA in a series of separate experiments. TMP, thymidine monophosphate; TDP, thymidine diphosphate; TTP, thymidine triphosphate. For the key to symbols we the legend to Fig. 1.

DESTABILIZED

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DNA

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have found that approximately 90% of the acid-soluble radioactivity from L-strain cells pulsed with tritiated thymidine consists of thymidine triphosphate. The reason for the failure of much of this material to pass through the dialysis membrane is unknown; possibly it is bound to a larger substance, and dissociates during hydroxyapatite chromatography.

4. Discussion These results indicate that the secondary structure of the newly replicated DNA differs from that of the previously existing DNA. Depending on the conditions of extraction, part of the newly replicated DNA can be isolated in either a single-stranded or double-stranded form. Our findings suggest that the newly formed DNA is not present in the cell as free single strands but rather in a unique destabilized state. The concept of a destabilized state is in some ways similar to the suggestion (Rosenberg & Cavalieri, 1964) that a substantial portion (up to 18%) of the template DNA ahead of the replication point exists in a pre-replicative “metastable state” and can be isolated as single strands by certain methods of extraction. However, our single-stranded fraction is almost entirely derived from newly synthesized DNA and in most instances contains only a negligible fraction (0 to 2%) of the total DNA as measured by distribution of 14C-labeled DNA. Painter & Schaefer (1969) have recently reported that a substantial portion of the newly replicated HeLa DNA extracted from cells in 1% sodium dodecyl sulfate by rapid freeze-thaw cycles (- 80 to SO’%)is single-stranded while Tsukada et al. (1968) found the newly replicated DNA extracted from rat liver nuclei at 0°C was entirely double stranded. This apparent contradiction is resolved by our findings which relate the isolation of newly replicated mammalian DNA in the single-stranded form to the temperature and method of extraction. Newly synthesized DNA of bacteria (Okazaki et al., 1968; Oishi, 1968) has been isolated as single strands, even when extracted under conditions where our HeLa DNA is completely double stranded. This may reflect a difference in the susceptibility of the strands of newly replicated DNA to separate during extraction, possibly due to fundamental differences in the replication process, such as involvement of histones in the mammalian chromosome. However, the extraction of newly replicated bacterial DNA in the double-stranded form by Yudelevich et al. (1968) suggests that a destabilized state may be involved in bacterial as well as in mammalian DNA synthesis. The molecular basis for the unique properties of newly replicated DNA is as yet unknown. It is conceivable that it exists within the cell as normal double-stranded DNA but has a lowered denaturation temperature as a result of specific chemical or structural factors. Pertinent to this are the findings of Felsenfeld, Sandeen $ von Hippel (1963) that RNase brings about a marked destabilization of the double helix and their suggestion that other proteins may have similar effects. Rosenberg & Cavalieri (1968) have discussed the role of shearing forces in denaturation and have suggested that the growing point may be attached to some large subcellular structure such as the cell membrane in such a way as to increase greatly the sensitivity of the newly synthesized DNA to denaturation by shearing forces that occur during extraction. We have, however, extracted newly replicated DNA in single-stranded form at more than 70°C below the normal denaturation temperature, a shift very much larger than any observed by either Felsenfeld et al. (1963) or Rosenberg & Cavalieri (1968). Another possibility, particularly in the light of the current concepts regarding the

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unwinding and rewinding of the helix during replication, is that the newly replicated DNA helix either exists in an extended form lacking the stabilizing stacking forces of the normal double helical molecule or is incompletely hydrogen bonded. The latter possibility implies either that loosening of the hydrogen bonding occurs between the template and newly synthesized strand following complementary base pairing or, alternatively, that base pairing between template and newly assembled nucleotides does not occur directly through hydrogen bonds but through some intermediate, such as an enzyme, and the newly synthesized daughter strand forms a hydrogen-bonded double helix only after a segment of single-stranded DNA is synthesized (cf. discussions by Paoletti et al., 1967 and Oishi, 1968). Our observations seem to be more compatible with this model. It is possible that attachment to membranes or association with specific proteins may be involved in maintenance of this incompletely hydrogen bonded or destabilized state within the nucleus. In our experiments to date it appears that disruption of the nuclear structure in the presence of the deproteinizing reagent (sodium para-amino salicylate) is the essential step in the preferential conversion of newly replicated DNA to the single-stranded form. We thank Dr Frank Defilippee for the sampleof Non-Id& P40 detergent and for his many helpful suggestions regarding its use and Dr Florence K. Millar for her assistance in computer analysis of the data. REFERENCES Bernardi, G. (1906). Nature, 206, 779. Berns, K. I. t Thomas, C. A. Jr. (1965). J. Mol. Biol. 11, 476. Cairns, J. (1966). J. Mol. Bid. 15, 372. Cleaver, J. E. t Holford, R. M. (1965). B&him. biophys. Acta, 103, 654. Eagle, H. (1959). Science, 180, 432. Felsenfeld, G., Sandeen, 0. & von Hippel, P. H. (1963). Proc. Ncrt. Ad. Sci., Wad.

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