Early- and late-replicating deoxyribonucleic acid complexes in HeLa nuclei

108

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 95294

EARLY- AND L A T E - R E P L I C A T I N G DEOXYRIBONUCLEIC ACID COMPLEXES IN HELA NUCLEI

GERALD C. M U E L L E R AND KAZUTO K A J I W A R A

McArdle Laboratory, University o/ Wisconsin, Madison, Wisc. (U.S.A.) (Received March, 23rd, 1965)

SUMMARY

The DNA which replicated early in the period of DNA synthesis in synchronized cultures of HeLa cells was labeled with [3H~thymidine. After several generations of random growth the cells were resynchronized and exposed to bromodeoxyuridine for various portions of the DNA-synthesis period. The aH label was carried in the bromodeoxyuridine-containing, heavy hybrid of the DNA that replicated early in the second synchronized cell cycle.

INTRODUCTION

Autoradiographic and kinetic studies of animal and plant cells have revealed the focalized nature of DNA synthesis within a nucleus. The process appears to involve many discrete DNA units which are made at different times and are distributed non-randomly among chromosomes and along individual ones 1-4. Using HeLa cultures synchronized by the induction and reversal of a thymidineless state, it was shown that early-labeling DNA units are ready to replicate on reversal of the thymidineless state 5. In contrast, DNA synthesis in the late-labeling units depends on metabolic processes which can be inhibited by actinomycin D and puromycin ~,6. An additional distinction between the DNA fractions has been found: incorporation of BdeU into DNA complexes made early in the period of DNA synthesis greatly decreased cloning ability, whereas BdeU incorporated into late-labeling DNA complexes had little effectL These experiments imply that synthesis of DNA in the nucleus is a temporally ordered process and that early- and late-replicating DNA complexes contain distinct genetic information. The present experiments with synchronized cultures show biophysically that DNA units identified by the time of replication in one cell generation retain their characteristic replication sequence in subsequent generations. A preliminary report of this work has appeared elsewhere 8. Abbreviations: BEH-medium, Eagle's HeLa medium containing io °/o bovine serum, o.i mM serine, o.I mM glycine, and o.oi mM inositol; BdeU, 5-bromodeoxyuridine; deT, deoxyribosylthymine.

Biochim. Biophys. Acta, 114 (1966) lO8-115

REPLICATION SEQUENCE OF H e L a

DNA

lO 9

METHODS

Culture o/ HeLa cells The origin and maintenance of the H e L a cell strain has been described in detail previously 9. Monolayer cultures in 32-oz p h a r m a c y bottles were prepared b y inoculating approx. 5" IOe cells in IOO ml BEH-medium; spinner cultures were made at the same cell concentration in spinner medium (BEH-medium, Ca *+ and Mg *+ omitted, supplemented with o.I % pluronic F-68) (ref. IO). Incubation and manipulations were carried out at 37 ° .

Synchronization o/ cultures DNA synthesis in the cultures was synchronized b y adding amethopterin (I/~M) and adenosine (5°/~M) 24-3 ° h after inoculation to block the endogenous synthesis of thymidine (deT)e, 9. 16 h later the thymidineless state was reversed b y adding IO/~M deT, [3HJdeT (2.5 mC/mmole, Schwarz BioResearch Inc.), E14C~deT (I mC/mmole or 5 mC/mmole, New England Nuclear Corporation) or IO #M of the bromine-containing analog BdeU (California Corporation for Biochemical Research) or [aHlBdeU (2.5 mC/mmole, Nuclear Chicago Corporation). Cultures so treated synthesize DNA in a synchronous wave that lasts about 6 h and approximately doubles the DNA of the culture e. This is followed b y a wave of mitosis in which the cell number nearly doubles.

Extraction o/ DNA Cells from monolayer cultures were freed b y a 5-min treatment with 2o ml of 0.05 % trypsin at 37 ° . Cells from such suspensions or from the spinner cultures were harvested b y centrifugation at low speed. The nucleic acids were isolated from the cell residues b y alternate extractions with phenol and a chloroform-isoamylalcohol mixture. In a typical extraction 2-lO 7 cells were suspended in 2. 7 ml of cold 0.2 M sodium citrate and were lysed during a Io-min period in the cold with 0.3 ml of 5 % sodium dodecylsulfate in 0.2 M sodium citrate. The lysate was shaken for I min on a Vortex mixer with 3.0 ml of 80 % phenol. After adding 0.3 ml of 5 M NaC1 in 0.2 M sodium citrate, the shaking was continued for I rain. The aqueous phase, obtained b y centrifuging 5 rain at IOOoorev./min (high-speed attachment, International centrifuge, model PR-2), was shaken for I rain with 3.0 ml of a 24:1 mixture of chloroform and isoamylalcohol and then was recentrifuged. The alternate extractions with phenol and chloroform-isoamylalcohol were repeated two more times. The mixed nucleic acids were precipitated with 9.0 ml of 95 % ethanol and dissolved in 2.0 ml of Tris-Mg ~+ buffer (0.02 M Tris-2 mM MgC12, p H 7-4).

Gradient centri[ugation o/ DNA To isolate the normal H e L a DNA or the heavy, BdeU-containing hybrid from the extract of nucleic acids, I.o-ml aliquots of the nucleic acid solution containing approx. IOO #g DNA were mixed with 3.0 g CsC1 and 1.2 ml water to give a density Biochim. Biophys. Acta, 95 (1966) lO8-115

iio

G.C. MUELLER, K. KAJIWARA

of 1.72 as determined by refractometry. A 2.5-ml aliquot was centrifuged at 2o ° for 65 h at 35 ooo rev./min in a Spinco SW-39 rotor. 5-drop fractions were collected and diluted with I.o.mLwater prior to reading the absorbance at 260 m# in a Beckman DU speetrophotometer. Radioactivity was determined by counting a I.o-ml aliquot in IO ml of BRAY'S solution 11 in a Packard Tricarb liquid scintillation spectrometer. The yield of DNA from BdeU-treated cells was usually slightly lower than that from deT-treated cells. The reduced yield did not interfere with the experiments or alter substantially the results.

RESULTS

Incorpora~,on o/ ~deU into DNA and the separation o/normal and heavy DNA BdeU will substitute completely for deT in the reversal of the thymidineless state in HeLa cells~. The cells incorporate BdeU at a rate equivalent to the incorporation of deT, and BdeU-treated cells from synchronized cultures divide after completion of DNA synthesis in a manner similar to that of deT-reversed cultures 7. The DNA resulting from one cycle of replication with BdeU is a hybrid containing one strand of newly synthesized, BdeU-containing DNA and one strand of normal DNA which was the template 1.. A preliminary experiment was undertaken to verify that the newly synthesized DNA-hybrid containing BdeU could be isolated by centrifugation in a CsC1 gradient, as has been described 1.. Two monolayer cultures were prelabeled with EuC]deT. I day later [~H3deT was added to Culture A, and [3H~BdeU was added to Culture B for a 24-h labeling period. Amethopterin and adenosine were included in the media during the final 24 h to block endogenous synthesis of deT. It was expected that one culture would contain heavy DNA with 14C from the first synthesis period and aH from the second; the other culture would contain DNA of normal weight also labeled with 14C and 3H. The nucleic acids from the two cultures were extracted and centrifuged separately in CsC1. In both cases I4C of the prelabeled DNA and ~H of the newly synthesized DNA centrifuged in the same peaks (Fig. I). Due to its higher density, the DNA from the BdeU-treated cells sedimented further into the gradient (Tubes 20-25, Fig. IB) than the normal DNA (Tubes 32-38, Fig. IA). The difference in density of the normal and BdeU-hybrid of DNA was sufficient to separate them in future, combined experiments. A small amount of normal DNA was observed in the nucleic acid patterns from the BdeU-treated cells (Tubes 35-40, Fig. IB); it came from cells that did not replicate DNA in the second synthetic period. If cultures were exposed to BdeU for only part of a synchronized period of DNA synthesis, two, well separated peaks were obtained; the denser contained BdeU. Fig. 2 illustrates this fact.

Sequential replication o / D N A in synchronized cultures o/ HeLa cells The characteristic patterns of chromosomes in autoradiographs of pulselabeled cells1--* imply that the DNA units along the chromosomes are replicated in a temporal sequence which is repeated in each reproductive cycle. If true, it should be demonstrable that DNA synthesized either early or late in one cycle is replicated Biochim. Biophys. Acta, 116 (1966) lO8-i1.5

REPLICATION SEQUENCE OF

0.5

A

[14C],:I¢'I"AND

HeLa DNA

[3H]clcT

0.4 ::L 0E 0.3 (40 (Xl

zlz

5

i! ~-~

.0.4

BdeU 3-6h

C

BdeU 0 - 6 h

IN

'~ 0 2

o

0.3

B

Q2

38

0.1

BdeU 0 - 3

0.4

4

"~ 0.2

A

O.4

0.2

02

0.1

O' I0 20 30 40 50 FRACTION NUMBER

I0 20 30 40 50 FRACTION NUMBER

Fig. I. Patterns of normal DNA and DNA containing I3deU after density gradient(centrifugation in CsC1. Two logarithmically growing monolayer cultures of 5' lO6 cells in BEH-medium were prelabeled for one generation (24 h) with 8/tM [14C]deT (o.8/~C). The media were replaced with fresh B E H - m e d i u m containing i #M amethopterin and 5o/zM adenosine. Either [3H]deT (io/zM, 2.5/zC, A) or [3H]BdeU (lOb*M, 2.5/zC, B) was added. After 24 h the ceils (8. lO7) were harvested and the nucleic acids extracted. Aliquots of approx, ioo/tg DNA were centrifuged in CsC1, density 1.72, for 65 h at 35 ooo rev./min in a SW-39 rotor. 5-drop fractions were collected; their absorbance at 260 m/z ( G - C ) ) and counts/min per fraction of SH (C)-C)) and 1'C ( O - O ) were determined. Fig. 2. Separation of early- and late-replicating DNA from synchronized cultures as the BdeUhybrid. 1. 3. lO7 cells in ioo ml spinner medium were synchronized by treatment for 16 h with amethopterin and adenosine. At t h a t time []*C]deT (2 #M, i #C) was added for 6 h. The medium was replaced with 300 ml spinner medium containing 5° $zM adenosine and 14/~M nonradioactive deT. After 79 h of random growth the culture was divided in three portions each containing 3" lO7 cells. A was exposed to 8.2 #M BdeU from o to 3 h, B to BdeU from 3 to 6 h, and C to BdeU from o to 6 h; io #M deT was present during the intervals when BdeU was not. The nucleic acids were extracted, separated, collected, and analysed as described before. Radioactivity data appear in Table II.

during the same period of subsequent cycles. To test this concept, cultures of HeLa cells were synchronized by amethopterin treatment, and the DNA made during the first part of the period of synchronous DNA synthesis was labeled with 14C. The cells were permitted to grow randomly for several generations, whereupon they were synchronized a second time. Replicate cultures were exposed to BdeU during different portions of the DNA-synthesis period (Fig. 3). The heavy DNA that contained BdeU was separated from the normal DNA by density gradient sedimentation in CsC1, and both fractions were assayed for the amount of radioactivity which was carried. In a typical experiment spinner cultures containing 7.5.1o 6 HeLa cells in IOO ml of medium were grown logarithmically for 30 h, then the thymidineless state Biochim. Biophys. Acta, 114 (1966) i o 8 - 1 i 5

112

G. C~ MUELLER, K. KAJIWARA J, F i r s t Synchronized DNA Synthesis J

Cul t ure

0

Amet hopter i n

[ 14c ]deT

30

53h

I Second Synchronized DRA Synthesis I Amethopter i n & Adenosine

,l

Reversal, Culture divided

Cells harvested

42

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BdeU

B

BdeU

)) )deT

C de'l'.~).BdeU

~,deT , )I I

O deT

l

)BdeU

2

3

4

5

. )i

6

7h

Fig. 3, Experimental protocol for double-labeling of early-replicating DNA

of HeLa cel|s. The

results are given in Fig. 4 and Table I.

was induced with amethopterin and adenosine. After 16 h [14C]deT was added to reverse the deficiency state and initiate synchronous replication of DNA. 2 h later, after the replication of approx. 25 % of the DNA in the culture, the medium was replaced with one containing nonradioactive deT and adenosine. Accordingly, early-replicating DNA would be radioactive, while the remainder of the complement of DNA in each cell would be synthesized from nonradioactive deT. The cells were grown randomly for 2 days. The 4" lO7 cells and 400 ml of fresh medium were transferred to a large spinner flask. 30 h later the cells were resynchronized b y the addition of amethopterin and adenosine. After 16 h of the thymidineless state the culture was divided into four equal amounts (Subcultures A, ]3, C, and D) in small spinner flasks. Culture A was incubated with BdeU during a 7-h period of DNA synthesis. Culture B was reversed with BdeU, removed from that medium after 3 h, and incubated with deT for the rest of the experiment. The cells of Culture C were incubated in medium containing deT for the first 2 h, in medium containing BdeU for the next 3 h (from 2 to 5 h after reversal of the thymidineless state), and then were returned to medium containing deT for the last 2 h. The cells in Culture D were incubated with deT for the first 4 h and then were transferred to BdeU for the final 3 h. The cells were harvested 7 h after the reversal of the thymidineless state and the nucleic acids were extracted. Aliquots of the nucleic acids were centrifuged in a CsC1 gradient and the absorbance of the fractions determined. Fig. 4. shows the density patterns of the nucleic acids from the four subcultures. The area under the heavy, BdeU-containing peak of DNA (Tubes 18-26) represents Biochim. Biophys. Acta, 114 (1966) lO8-115

REPLICATION SEQUENCE OF H e L a

0.3

_A

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Fig. 4. The isolation of D N A s y n t h e s i z e d d u r i n g different intervals in synchronized culture. Fig. 3 s h o w s t h e sequence of e x p e r i m e n t a l procedures. Cell n u m b e r s and v o l u m e of s p i n n e r med i u m : at o h, 7.5' IOe cells in ioo ml; at 96 h, 4" lOT cells in 40o ml; 126 h, 8. lO T cells in 4o0 ml; at 149 h, 2.5" lO T cells A, 2.5" 1o~ cells ]3, 2. 7. lO T cells C, 2. 7. IO~ cells D in ioo ml. Concentrations of c o m p o u n d s added: i / , M a m e t h o p t e r i n ; 5 ° #M adenosine; 1.2/~M [l~C]deT, o.6/*C; 20 FM deT, first synchronized s y n t h e s i s period; 5/~M deT, second synchronized synthesis period; 5/~M BdeU. The D N A was extracted, centrifuged, collected, and analysed as described before. Radioa c t i v i t y d a t a a p p e a r in Table I.

the portion of the total DNA of the culture which was replicated during the time of contact with BdeU. The peak of normal DNA (Tubes 33-4 I) comprised DNA synthesized before or after incubation with BdeU. It also included DNA from cells which did not replicate during the test period. The heavy DNA and normal DNA fractions were dialysed against 2000 ml distilled water for 16 h, and the absorbance and radioactivity of each were determined. The radioactivity in the BdeU-DNA is derived in all cases from the DNA labeled early in synthesis period of first synchronization. Comparing the specific activities of these fractions shows that the 14C was carried preferentially in the heavy BdeU-DNA made during the first 3 h of DNA synthesis (Table I). The lowest specific

TABLE I SPECIFIC ACTIVITY OF BdeU-CONTAINING AND NORMAL H e L a DNA: EARLY-REPLICATING D N A PR~LABELED WITH l~C Figs. 3 and 4 give the e x p e r i m e n t a l details. The h e a v y D N A fraction was pooled from T u b e s 18-24, the n o r m a l from T u b e s 33-4 o. B o t h were dialyzed against 2ooo ml w a t e r for 16 h before assaying. The relative specific activities of the h e a v y DNA, if A = i.oo, are B, 1.74; C, o.71; D, o.55. Culture

A B C D

Interval with B d e U (h )

I~g

Heavy D N A

counts/min "ttg I~g

Normal D N A counts /min . #g

o-7 o- 3 2-5 4-7

49.4 18.8 24.3 20.6

12.6 22.o 9.0 6.9

23.2 12.1 19. i 21.6

22.6 47.6 40.4 49.o

Biochim. Biophys. Acta, 116 (1966) lO8-115

II4

G.C.

MUELLER,

K.

KAJIWARA

activity appeared in the BdeU-containing fraction replicated at the end of tile second synthesis period. DNA of normal density in Culture A derives from those cells which failed to replicate their DNA during the second synchronized synthesis period; therefore its specific activity is high. Ideally, this DNA would have a specific activity two times that of the DNA containing BdeU. DNA of normal density from Cultures B, C, and D includes this fraction; consequently their specific activities are higher than they would be if all the DNA of the culture had replicated. Taking this into account, it is apparent that the reciprocal relationship of the specific activities of the DNA's of normal density confirms the findings of the BdeU-containing fraction. In similar experiments [14CldeT was introduced into the late-replicating DNA units during the first synchronization. The subsequent resynchronization and fractional synthesis of the DNA with BdeU were carried out as described above. The results of the experiments are in agreement with those labeling the earlyreplicating DNA: the late-replicating DNA of one cycle also replicates late in the DNA synthesis period of subsequent cycles. Table lI shows the results of a companion experiment designed to test whether BdeU selects DNA of a particular composition instead of following an ordered TABLE II S P E C I F I C A C T I V I T Y OF NORMAL A N D B d e U - C O N T A I N I N G

DNA P R E L A B E L E D W I T H I~C

Fig. 2 gives the experimental details. The heavy DNA fraction was pooled from Tubes 16-21, the normal from Tubes 31-38; these were dialysed against 2ooo ml water for 16 h before assaying. The lmtative specific activities of the heavy DNA, if C ~ i.oo, are A, o.91; B, o.9o.

Cuttu~¢

A B C

Interval with BdeU (h)

I~g

Heavy D N A

counts/rein'lag I~g

Normal D N A counts]min" t*g

o- 3 3-6 0-6

37.8 36.6 73.2

38.2 37.9 41.9

57.2 56.9 82.0

94.6 98.3 4°.8

sequence of replication. Cells were prelabeled with [14CldeT for 6 h during the first period of synchronized synthesis. After 66 h of random growth the cells were resynchronized and exposed to BdeU for a 6-h period or for the first or last 3 h of this period. The nearly identical specific activities of the heavy DNA from Cultures A, B, and C show that BdeU does not discriminate between DNA molecules of different compositions.

DISCUSSION

Our findings illustrate that the DNA molecules made during one part of the DNA-synthesis period are replicated in the same temporal relationship to other DNA molecules in subsequent periods of DNA synthesis of daughter cells. This clearly provides a molecular basis for the earlier autoradiographic studies showing the focalized and specific patterns of DNA synthesis of individual mammalian chromosomes 1~. Evidently the replication of DNA within a chromosomal subunit is regulatBiochim. Biophys. Acta, 114 (1966) lO8-115

REPLICATION SEQUENCE OF H e L a

DNA

II5

ed b y m o l e c u l a r e v e n t s occurring a t t h a t site. BRAUN et al. 13 h a v e shown receaatty t h a t the sequence of D N A r e p l i c a t i o n in the slime m o l d P h y s a r u m p o l y c e p h a l u m is also t e m p o r a l l y ordered. Our earlier o b s e r v a t i o n s t h a t a c t i n o m y c i n D a n d p u r o m y c i n p r e v e n t t h e s y n t h e s i s of t h e l a t e - r e p l i c a t i n g DNAS, s i m p l y t h a t synthesis of b o t h new R N A a n d new p r o t e i n is n e c e s s a r y for this p h e n o m e n o n . This is in accord ~ studies of LARK a n d associatesS4,15 on t h e replication of the b a c t e r i a l c h r o m o s o m e s w h i c h show the d e p e n d e n c e of this process u p o n p r o t e i n synthesis. The correlation of l a t e labeling D N A w i t h h e t e r o c h r o m a t i c areas of chromosomes 1,is suggests t h e involvem e n t of the n e w l y s y n t h e s i z e d p r o t e i n in b o t h t h e i n i t i a t i o n of D N A synthesis a n d t h e r e p r o d u c t i o n of t h e h e t e r o c h r o m a t i c c h a r a c t e r a t such sites. Since R N A synthesis in h e t e r o c h r o m a t i e areas is less active t h a n in the e u c h r o m a t i n l L 18, t h e genetic expression of l a t e - l a b e l i n g D N A m a y be limited. I n a g r e e m e n t w i t h this concept, t h e i n c o r p o r a t i o n of B d e U into l a t e - r e p l i c a t i n g D N A has less effect on t h e s u r v i v a l of cells t h a n its i n t r o d u c t i o n into e a r l y - r e p l i c a t i n g D N A L T h e d a t a s u p p o r t t h e conclusion t h a t t h e o r d e r e d s e q u e n c e of D N A s y n t h e s i s results from t h e existence of at least two different p h y s i c a l associations of D N A w i t h i n t h e c o m p l e x s t r u c t u r e of t h e m a m m a l i a n chromosome. T h e replication of t h e D N A in these associations has different m e t a b o l i c prerequisites which can be dist i n g u i s h e d e x p e r i m e n t a l l y . S y n c h r o n i z e d cultures p r o v i d e the o p p o r t u n i t y to isolate a n d c h a r a c t e r i z e t h e D N A ' s a n d a n y R N A a n d p r o t e i n affecting t h e m .

ACKNOWLEDGEMENTS

The a u t h o r s are g r a t e f u l for the excellent assistance of Mrs. K. DEIGHTON a n d Mrs. E. ERIKSON in t h e c o n d u c t of these e x p e r i m e n t s a n d also acknowledge t h e assistance of Mrs. H. H. BALDWIN in t h e p r e p a r a t i o n of t h e m a n u s c r i p t . T h e w o r k was s u p p o r t e d b y a U.S. P u b l i c H e a l t h Service G r a n t CA-o7175 from t h e N a t i o n a l Cancer I n s t i t u t e . G.C.M. is a recipient of a research carreer award, U.S. P u b l i c H e a l t h Service. REFERENCES I 2 3 4 5 6 7 8 9 io ii 12 13 14 15 16

17 18

J. H. TAYLOR, J. Biophys. Biochem. Cytol., 7 (196o) 455. E. STUBBLEFIELDAND G. C. MUELLER, Cancer Res., 22 (1962) IO91. J- GERMAN, J. Cell Biol., 2o (1964) 37. T. C. Hsu, J. Cell Biol., 23 (1964) 53. G. C. MUELLER, Exptl. Cell Res., Suppl., 9 (1963) 144G. C. MUELLER, I{. KAJIW'ARA, E. STUBBLEFIELD AND R. R. RUECKERT, Cancer Res., 22 (1962) lO84 . K. KAJIWARA ANn G. C. MUELLER, Biochim, Biophys. Acta, 9I (1964) 486. K. KAJIWARA AND G. C. MUELLI~R, Proe. Am. Assoc. Cancer Res., 5 (I964) 33. R. R. RUECKERT AND G. C. MUELLER, Cancer Res., 20 (196o) 1584 . l-I. E. SWIM AND R. V. PARKER, Proc. Soc. Exptl. Biol. Med., lO3 (196o) 252. G. A. BRAY, Anal. Biochem., i (196o) 279. E. H. SIMON, J. Mol. Biol., 3 (1961) IOI. R. BRAUN, C. MITTERMAYERAND H. P. RUSCH, Proc. Natl. Acad. Sci. U.S., 53 (1965) 924. K, G. LARK, T. REPKO ANn E. J. HOFFMAN,Biochim. Biophys. Acta, 76 (1963) 9C. LARK AND K. G. LARK, J. Mol. Biol., IO (1964) 12o. A. LIMA-DE-FARIA, J. Biophys. Biochem. Cytol., 6 (1959) 457. T. C. Hsu, Exptl. Cell Res., 27 (1962) 332. L. BERLOWlTZ, Proc. Natl. Acad. Sci. U.S., 53 (1965) 68. Bioehim. Biophys. Acta, 114 (1966) lO8-115