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36 Biochimica et Biophysica Acta, 447 (1976) 36--44 © Elsevier/North-Holland Biomedical Press BBA 98692 CHAIN ELONGATION AND JOINING OF DNA SYNTHESIZED DURING HYDROXYUREA TREATMENT OF CHINESE HAMSTER CELLS R.A. WALTERS, R.A. TOBEY and C.E. HILDEBRAND Cellular and Molecular Biology Group, Los Aiamos Scientific Laboratory, University of California, Los Alamos, N.M. 87545 (U.S.A.) (Received January 16th, 1976) (Revised manuscript received April 18th, 1976) Summary We have previously presented evidence that hydroxyurea treatment of syn- chronized G1 Chinese hamster cells did not prevent the entry of cells into the DNA synthetic period but that the DNA synthesized during this period (in which total DNA synthesis was severely depressed) was quite small (Wa!ters, R.A., Tobey, R.A. and Hildebrand, C.E. {1976) Biochem. Biophys. Res. Com. 69, 212--217). In view of the reported effects of hydroxyurea on deoxyribo- nucleoside metabolism and possible relationship to control of DNA replication (Bjursell, G. and Reichard, P. (1973) J. Biol. Chem. 248, 3904--3909 and Wai- ters, R.A., Tobey, R.A. and Ratliff, R.L. (1973) Biochim. Biophys. Acta 319, 336--347), we examined the fate of DNA synthesized during and shortly after hydroxyurea treatment to determine if this DNA exhibited any kinetic behav- ior which might be an indicator of aberrant synthesis. We found that, upon hydroxyurea removal, DNA grew at a linear rate of 0.98 -+ 0.12 • 106 dalton/ min (0.98 + 0.12 pm/min) for about 2.3 h. Beginning at 2.3 h, DNA with a molecular weight approx. 1.4 • 108 was very rapidly integrated into bulk DNA of ~> 3.5 • 108 daltons. The apparent growth rate of the 1.4 • 108 dalton DNA was approx. 10.6 pm/min. The data suggest that, at least for this DNA, joining into bulk DNA required one-third to one-half of the S period to begin and, once begun, occurred very rapidly. The possibility of integration of replicon clusters is considered. Introduction In mammalian cells, DNA replication occurs on individual units (replicons) and proceeds bidirectionally in most of the replicons [4--6]. The size of Chi- Abbreviations: the terms G1, S, and G 2 denote the phases of the mammalian cell cycle: G 1 is the period from the end of mitosis to the DNA synthetic phase (S), and G 2 is the time period from the end of the DNA synthetic phase to initiation of mitosi~

Chain elongation and joining of DNA synthesized during hydroxyurea treatment of Chinese hamster cells

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Biochimica et Biophysica Acta, 4 4 7 ( 1 9 7 6 ) 3 6 - - 4 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l P r e s s

B B A 9 8 6 9 2

CHAIN ELONGATION AND JOINING OF DNA SYNTHESIZED DURING H Y D R O X Y U R E A TREATMENT OF CHINESE HAMSTER CELLS

R . A . W A L T E R S , R . A . T O B E Y a n d C.E. H I L D E B R A N D

Cellular and Molecular Biology Group, Los Aiamos Scientific Laboratory, University o f California, Los Alamos, N.M. 87545 (U.S.A.)

( R e c e i v e d J a n u a r y 1 6 t h , 1 9 7 6 ) ( R e v i s e d m a n u s c r i p t r e c e i v e d Apr i l 1 8 t h , 1 9 7 6 )

Summary

We have previously presented evidence that hydroxyurea t reatment of syn- chronized G1 Chinese hamster cells did not prevent the entry of cells into the DNA synthetic period but that the DNA synthesized during this period (in which total DNA synthesis was severely depressed) was quite small (Wa!ters, R.A., Tobey, R.A. and Hildebrand, C.E. {1976) Biochem. Biophys. Res. Com. 69, 212--217). In view of the reported effects of hydroxyurea on deoxyribo- nucleoside metabolism and possible relationship to control of DNA replication (Bjursell, G. and Reichard, P. (1973) J. Biol. Chem. 248, 3904--3909 and Wai- ters, R.A., Tobey, R.A. and Ratliff, R.L. (1973) Biochim. Biophys. Acta 319, 336--347), we examined the fate of DNA synthesized during and shortly after hydroxyurea t reatment to determine if this DNA exhibited any kinetic behav- ior which might be an indicator of aberrant synthesis. We found that, upon hydroxyurea removal, DNA grew at a linear rate of 0.98 -+ 0.12 • 106 dal ton/ min (0.98 + 0.12 pm/min) for about 2.3 h. Beginning at 2.3 h, DNA with a molecular weight approx. 1.4 • 108 was very rapidly integrated into bulk DNA of ~> 3.5 • 108 daltons. The apparent growth rate of the 1.4 • 108 dalton DNA was approx. 10.6 pm/min. The data suggest that, at least for this DNA, joining into bulk DNA required one-third to one-half of the S period to begin and, once begun, occurred very rapidly. The possibility of integration of replicon clusters is considered.

Introduction

In mammalian cells, DNA replication occurs on individual units (replicons) and proceeds bidirectionally in most of the replicons [4--6] . The size of Chi-

Abbreviations: the t e r m s G1, S, and G 2 d e n o t e the phases o f the mammal i an cell cycle: G 1 is the per iod f r o m the end o f m i t o s i s to the D N A syn th e t i c phase (S), and G 2 is the t ime period f rom the

end of the DNA synthet ic phase to init iation of mitosi~

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nese hamster replicons can very from a few microns to over 100 pro, with an average replicon size of approx. 30 pm [4]. Both the number and replication rate of replicons can vary throughout the DNA synthetic period [6,7], and at least some replicons appear to replicate in clusters [5]. Owing to these com- plexities of DNA replication, it is difficult to measure the true replication rates of individual replicons other than by DNA fiber autoradiography. However, this technique is also limited in that it is difficult to examine the overall organiza- tion of replication and the integration of newly synthesized DNA into high molecular weight DNA. While isokinetic density gradient sedimentation cannot be used easily to estimate the rate of replication of individual replicons, it can be used to obtain information concerning the integration of newly synthesized DNA into high molecular weight (or bulk) DNA. Even in this case, however, care must be used in interpreting the results, since (1) even short radioactive pulses will yield DNA distributed from relatively low to quite high molecular weights owing to the fact that, during the pulse, some replicons will initiate synthesis while others will continue and/or terminate synthesis; (2)radioactive labels require a significant period of t ime to chase from the deoxyribonucleo- side triphosphate pools; and (3) the kinetics of chase of newly replicated DNA into bulk DNA may very well depend upon the method of isolation of bulk DNA, the size of which will vary greatly [8--10].

We report here results obtained from a study of the integration into bulk DNA of relatively small DNA synthesized early in the DNA synthetic phase of Chinese hamster cells. We have employed a protocol which enables us to exam- ine the growth across a wide molecular weight range of a population of initially small DNA and the subsequent integration of this DNA into bulk DNA, the size of which is in the molecular weight range of the largest DNAs that can be de- monstrated unambiguously to be single-stranded upon alkaline sucrose density gradient centrifugation [ 11]. We were interested in this system for a number of reasons. Hydroxyurea t reatment permits the synthesis of small DNAs in S phase cells of HEp2 [12], L [13], and polyoma-infected cells [14] and appar- ently allows GI Chinese hamster cells to enter the DNA synthetic period and to initiate at least a limited number of rephcons [1]. Hydroxyurea t rea tment also induces a state in which purine deoxyribonucleoside triphosphates, rather than pyrimidine deoxyribonucleoside triphosphates, are in limiting concentrations [3,15]. Since it has been postulated that the dCTP pool may exert a controlling influence on DNA replication [2], it is possible that, under hydroxyurea block- ade, the small DNA synthesized might represent a replication intermediate. In addition, hydroxyurea t rea tment of synchronized G1 cells represents one method of obtaining a very highly synchronized cell population [16]. Since cells can initiate DNA replicon synthesis in the presence of hydroxyurea , infor- mat ion concerning the fate of this DNA would be useful.

Experimental

Chinese hamster cells (line CHO) in suspension culture were maintained free of Mycoplasma, as assayed by the method of House and Waddell [17], in F-10 * medium (Schwarz/Mann) supplemented with 15% newborn calf serum,

* F - I O d e n o t e s t h e n u t r i e n t m i x t u r e u s e d in p r o p a g a t i o n o f t h e c e l l s

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penicillin, and streptomycin. Cell concentrat ion was determined with an elec- tronic particle counter.

Cells were synchronized by selective detachment of mitotic cells from mono- layer cultures on glass and treated with hydroxyurea (1 mM) according to the protocol described by Tobey and Crissman [16]. The fraction of cells in the DNA synthetic period (S) was determined autoradiographically, as previously described [18].

Cellular DNA was labeled with either [Me-3H]thymidine (55 Ci/mmol, New England Nuclear) or [Me-14C]thymidine (55.3 Ci/mmol, Schwarz/Mann) prior to analysis by alkaline sucrose density gradient centrifugation. The details of gradient content and formation, calibration, and cell lysis (2 • 10 s cells per gra- dient} were as previously described [9]. Gradients were fractionated from the top, and fractions (1.2 ml) were collected onto glass fiber filters (Millipore No. AP2504200). Prior to use, 10 #g single-strand DNA had been added to each filter, and the filters were soaked in 10% trichloroacetic acid and dried at room temperature. After gradient fractionation, the filters were batch-washed at 4°C five times with 5% trichloroacetic acid (34 filters per batch). The filters were then washed individually (under suction) six times with cold absolute ethanol, air dried, and counted as previously described [ 19,20].

Results

To examine the DNA synthesized during hydroxyurea t reatment (and im- mediately thereafter), the following protocol was employed. Hydroxyurea (1 mM) and 100 #Ci/ml [3H]thymidine were added 1 h after mitot ic synchro- nization. At 9.5 h after mitotic synchronization, the cells were removed from hydroxyurea and [3H]thymidine by low-speed centrifugation, washed twice

o ×

% x c~ 4

2 v

I I I I

L J I I 0 1 2 3 4

H o u r s o f t . e r r e m o v a l of h y d r o x y u r e o

Fig. 1. Kinet i c s o f [ 3 H ] t h y m i d i n e i n c o r p o r a t i o n into D N A . A l i q u o t s were r e m o v e d f r o m the c u l t u r e im- m e d i a t e l y prior t o and at s e l ec t ed intervals af ter r e m o v a l o f [ 3 H ] t h y m i d i n e and h y d r o x y u r e a . H y d r o x y - u r e a a n d [ 3 H ] t h y m i d i n e w e r e added to m i t o t i c a l l y s e l e c t e d ce l l s at I h p o s t - s y n c h r o n i z a t i o n and r e m o v e d

at 9 . 5 h p o s t - s y n c h r o n i z a t i o n .

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with cold F-10 culture medium, and resuspended in warm, fresh culture medi- um. At the time of hydroxyurea removal, 50--55% of the cells had entered S, as determined by autoradiography. At selected intervals after hydroxyurea and [3H]thymidine removal, aliquots were taken from the culture vessel for (1) de- termination of total radioactive DNA and (2) DNA analysis by alkaline sucrose density gradient centrifugation.

The data in Fig. 1 show that radioactive incorporation into DNA continued for 0.5 h into the chase period after removal of hydroxyurea and [3H]thymi- dine, during which time the radioactivity increased by 125%. This is not unex- pected, since (1) hydroxyurea-treated Chinese hamster cells accumulate dTTP at a faster rate than untreated cells [3] and (2) a minimum of 50% of the dTTP is derived from exogenous thymidine under our growth conditions [19,21]. From earlier work [3], we can estimate roughly the time that the dTTP acid- soluble pools could support DNA synthesis at the time of hydroxyurea remov- al. With approx. 130 pmol dTTP/106 cells, approx. 55% in S, and a 6-h S peri- od, enough dTTP exists to support DNA synthesis for 10--15 min. This would be a minimum estimate, since it assumes that the cell would transport no extra- cellular thymidine during this period. Thus, the 30-min interval required for label chase does not appear to be an unreasonable estimate.

I ~ r~ I I / I

_ q l l ., L ,' L $ \./ I, I l J - '

F r ~ c t l o n N o .

Fig. 2. A l k a l i n e s u c r o s e d e n s i t y g r a d i e n t p a t t e r n s o f D N A f r o m s y n c h r o n o u s cel ls a t s e l ec t ed t i m e s a f t e r r e m o v a l o f h y d r o x y u r e a a n d [ 3 H ] t h y m i d i n e . The p r o t o c o l o f s y n c h r o n i z a t i o n , 3H l abe l ing a n d h y d r o x y - u r e a t r e a t m e n t w a s t h e s a m e as in Fig . 1. (o o) [ 3 H ] D N A ; (v u) and ( . . . . . . ) [ 1 4 C ] D N A f r o m e x p o n e n t i a l l y g r o w i n g cel ls l a b e l e d f o r 3 6 h w i t h [ 1 4 C ] t h y m i d i n e . T h e d a s h e d l ines in B I H w e r e taken from pane l A. (A) 0 h chase ; (B) 0 . 2 5 h chase ; (C) 0 . 7 5 h c h a s e ; (D) 1 . 5 h c h a s e ; ( E ) 2 . 2 5 h chase ; (F ) 2 . 5 h chase ; (G) 2 . 7 5 h c h a s e ; a n d (H) 3 .0 h chase . G r a d i e n t s w e r e c e n t r i f u g e d in a n SW-27 r o t o r in a B e c k m a n L 3 - 5 0 ultracentrifuge for 6 . 2 5 h a t 1 5 ° C a n d 2 2 0 0 0 r e v . / m i n . The d irect ion o f sed imentat ion was l e f t t o r i gh t .

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4 0

The data in Fig. 2 show alkaline sucrose gradient patterns of DNA from cells harvested either immediately prior to (Fig. 2A) or at selected intervals after re- moval of hydroxyurea and [3H]thymidine (Fig. 2B--H). For comparison, the pattern of bulk DNA from cells labeled for 36 h during exponential growth with 0.05 pCi/ml [14C]thymidine is shown. It can be seen that the DNA syn- thesized prior to removal of hydroxyurea was quite small (< 1 • 107 daltons, Fig. 2A). During the chase period (Fig. 2B--H), the DNA grew progressively larger and, by 2.75 h, approx. 80% of the DNA sedimented with bulk DNA. Only at about 2.5 h into the chase period could we clearly discern major mul- tiple peaks of DNA (1.6 • 108 and 2.4 • 108 daltons, Fig. 2F) with molecular weights significantly less than that of bulk DNA. This does not mean that , in the size range examined here during chase (~< 1 • 107--3.5 • 108 daltons), other DNA intermediates of discrete molecular weights do not exist, but if they do, they cannot be resolved by the techniques used here. It should be noted from Fig. 2 that the longer chase times and fully labeled bulk DNA all yielded DNA sedimenting near the bo t tom of the centrifuge tube (e.g., fraction 29). This is a characteristic common to high molecular weight DNA in alkaline sucrose gra- dients, which we and others have attributed to wall effects (see ref. 9). No cor- rections have been made for this effect.

A summary of a number of experiments like the one shown in Fig. 2 examin- ing the growth of DNA after removal of hydroxyurea is presented in Fig. 3. The data obtained up to 2 h after hydroxyurea removal were plotted as the molecular weight at the peak of the DNA distribution and were fitted by linear regression analysis. A sample correlation coefficient of 0.985 was obtained for the data fit (e.g., 1.0 denotes a perfect fit), and the analysis was significant at

360

32C

28C ~-

o "o 24C .c

"~ 2 0 0

160

~ 120

8 0

4 0

o

I 1 L- 1 2 3

Hours after removal of hyclroxyurea

Fig. 3. Kinet i c s o f D N A chain g r o w t h in s y n c h r o n o u s ce l ia A t se l ec ted t i m e s after r e m o v a l o f h y d r o x y - u r ea and [ 3 H ] t h y m i d i n e , t h e D N A m o l e c u l a r w e i g h t at the p e a k w a s d e t e r m i n e d as in Fig . 2. The pro to - co l o f s y n c h r o n i z a t i o n , hyc troxyurea t r e a t m e n t , and [ 3 H ] t h y m i d i n e label ing wa s the same as in figs. 1 and 2. T h e m o l e c u l a r w e i g h t o f b u l k D N A w a s approx . 3 . 5 - 1 0 8.

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the 1% confidence level. The data indicate that DNA grew at a linear rate of 0.98 + 0.12 • 106 dalton/rain (or 0.98 + 0.12 pm/min assuming 1 pm = 106 daltons of single-strand DNA [22]) up to about 2.3 h after removal of hydroxy- urea. Beginning at 2.3 h, DNA with a molecular weight of approx. 1.4 • 108 was very rapidly integrated into bulk DNA of i> 3.5 • l 0 s daltons (see also Fig. 2E-- H). The apparent growth rate of 1.4 • l 0 s dalton DNA was approx. 10.6 pm/ min. Because of the very rapid nature of the process, we were not able, for technical reasons, to obtain additional points defining the rapid growth of 1.4 • 108 dalton DNA. Thus, the 10.6 pm/min apparent growth rate for this process was estimated by fitting by eye the data from 2.25 to 3.0 h after hydroxyurea removal, and the estimate must be considered to be only the best approxima- tion. (The data for the 2.5-h chase were also plotted as the molecular weight of the DNA peaks in the gradient. We have observed discontinuities at the 2.5-h time point in three separate experiments.) Even with this qualification, how- ever, we feel that the very rapid integration of the 1.4 • 108 dalton DNA is sig- nificant in that the rate is greater by far than estimates of the rate of fork movement in Chinese hamster cells [ 7].

It should be noted here that the DNA synthesized during hydroxyurea treat- ment does not appear to be repair replication. It has been shown that hydroxy- urea will not induce detectable DNA strand breaks in mammalian cells during a 24-h t reatment [12]. In addition, since repair replication is not inhibited by hydroxyurea [23], any [3H]thymidine incorporated as a consequence of repair synthesis should appear in the region of bulk DNA (t> 3.5 • 108 daltons). We find no evidence of such incorporation during hydroxyurea t reatment (Fig. 2A).

Discussion

As will be reported elsewhere [ 1 ], hydroxyurea treatment of mitotically syn- chronized G1 cells allowed the cells to enter the DNA synthetic period at the same time and rate as untreated controls, although the amount of DNA synthe- sized was severely depressed. While hydroxyurea t reatment of synchronized G, cells permits one to obtain a very highly synchronized cell population, the de- tails of which have been reported [ 16], the point of synchronization appears to be on the S side of the G, /S boundary. The DNA synthesized during hydroxy- urea t reatment exhibited a bimodal distribution and was smaller (Fig. 2A) than the average 30-#m replicon size, bu t at least some of the DNA was approaching the size of smaller 10-gm replicons reported for Chinese hamster cells (line CHO) [4]. Because of (1) the small amount of DNA synthesized [1], (2) the continuous presence of [3H]thymidine as cells traversed G, and entered S, (3) the large number of S-phase cells (50--55%), and (4) the high degree of syn- chrony achieved during hydroxyurea treatment, we presume the DNA synthe- sized during this period to be DNA synthesized in early S. The significance of the bimodal distribution has not been assessed at this time.

Within 0.25 h after removal of hydroxyurea and [3H]thymidine, radioactivi- ty in the DNA increased by about 60% (data not shown) and showed a maxi- mal 125% increase within 0.5 h (Fig. 1). However, neither the 0.25-h sample (Fig. 2B) nor the 0.5-h sample (gradient pattern not shown) had a large fraction of radioactivity in the top fractions. In fact, the 0.25-h sample (Fig. 2B) show-

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ed that the DNA had grown to approx. 1.8 • 107 daltons and that about 85% of the DNA sedimented in a well<lefined peak. This suggests that the residual in- corporation after removal of hydroxyurea and [3H]thymidine either (1) occur- red on replicons which had initiated DNA synthesis during hydroxyurea treat- ment or (2) occurred on replicons which initiated in a highly synchronous manner after hydroxyurea removal. While we cannot exclude the second possi- bility we favor the first possibility for the following reason. Had a large portion of the label been incorporated into newly initiated replicons, we might have expected (based on the findings of Housman and Huberman [7] for Chinese hamster cells in early S) a good deal of radioactivity distributed in DNA <~ 6 • 106 daltons (e.g., fractions 1--6). Whatever the case, however, it is clear that most of the DNA synthesized during hydroxyurea t reatment exhibited similar kinetic behavior to that synthesized within 0.5 h after hydroxyurea removal. Had the DNA synthesized during hydroxyurea t reatment failed to chase into high molecular weight DNA, approx. 44% of the radioactivity would have remained in the top ten fractions, a result inconsistent with the data in Fig. 2. Thus, it would appear that the small DNA synthesized during hydroxyurea treatment, during which time (depending on growth conditions) either dATP [1,3] or dATP and dGTP [15] were in limiting concentrations, could be elongated once hydroxyurea was removed. These results are reminis- cent of those obtained in polyoma-infected 3T6 mouse fibroblasts in which hydroxyurea t reatment resulted in an accumulation of small, polyoma-specific DNAs which are precursors of long DNA chains [14]. One wonders if possibly initiation can occur when pyrimidine deoxyribonucleoside triphosphates are in sufficient supply, while chain elongation is sensitive to limiting concentrations of the purine deoxyribonucleoside triphosphates.

Following removal of hydroxyurea and [3H]thymidine, the DNA grew pro- gressively larger. There was no clearcut indication of a discontnuous accumula- t ion of DNA intermediates with discrete molecular weights up to 2.25 h after removal of hydroxyurea (Fig. 2B--E), as judged by the failure to find clearly separated DNA peaks. However, there may be distinct DNA intermediates [24] in the low molecular weight regions (< 1 • 107) which we have not resolved under the radioactive labeling and centrifugation conditions reported here (see Fig. 2A). Additional experiments will be needed to clarify this point. However, we did detect two peaks of DNA with molecular weights significantly less than that of bulk DNA at 2.5 h after removal of hydroxyurea (1.6 • 108 and 2.4 • 108, Fig. 2F) which may reflect a discontinuity in chain growth beginning at this time. Huberman and Horowitz [8] and Friedman et al. [10] have also re- ported, for Chinese hamster cells (line CHO) and L cells, respectively, that strand growth was continuous for DNA < 25 S. However, neither of these two studies would have detected the discontinuities in the molecular weight range we noted at 2.5 h after release from hydroxyurea, since the bulk DNA of Huberman and Horowitz [8] was much smaller than that reported here, and L cell DNA of > 3--6 • 107 daltons of Friedman et al. [10] was transferred direct- ly into rapidly sedimenting aggregate material. It should be noted here that there is no guarantee tha t the DNA synthesized prior to or shortly after remov- al of hydroxyurea is similar in all respects to that synthesized during the re- mainder of S. It is possible that the properties of DNA initiation and chain

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elongation reported here are characteristic of a special class of DNA. As shown in Fig. 3, DNA grew at an apparent rate of 0.98 + 0.12 pm/min up

to 2 h after hydroxyurea removal. Although a 0.98 pm/min rate of DNA chain elongation is not an unreasonable result [5], a number of factors should be considered. Housman and Huberman [7] recently reported that the rate of fork movement in Chinese hamster cells (line CHO) in early S was only about 0.2 #m/min. If the DNA synthesized during and shortly after hydroxyurea treat- ment is characteristic of early S DNA, a 0.98 #m/min growth rate is higher than might be expected, even assuming two forks per replicon. The very rapid inte- gration of approx. 1.4 • 108 dalton DNA into bulk dize DNA (Fig. 3) is interest- ing. Since cell synchrony decays continuously with time after hydroxyurea re- moval, one can legitimately question whether such a rapid phenomenon should occur in vivo. However, we believe that this is a real physiological response and is no t generated by our method of analysis for the following reasons. Rotor speed dependence of sedimentation of quite large DNAs [25] should not ac- count for this effect, as we have reduced the rotor speeds to half that used in this study and have found that, while the shape of the distribution of bulk DNA can be altered, the molecular weight at the peak changes little if at all (unpublished observations). Further, Hand [5] has reported that, while varying in DNA as a whole, timing of initiation events and rates of fork movement are quite closely regulated within clusters of active replication units. Thus, there is precedent for believing that some processes, once begun, could display a high degree of temporal order despite a decay of synchronization in the cell popula- t ion as a whole.

Because of its very rapid nature (approx. 10.6 pm/min equivalent growth rate), we suggest that the size increase beginning about 2.3 h after hydroxyurea removal (e.g., one-third to one-half of the S period) represents a rapid joining process rather than actual replication. It is interesting that the rapid joining process should occur with DNA of about 1.4 • 108 daltons. One might expect that DNA chains would elongate until they reach the maximal size of a replicon and that then one would see DNA chains at adjacent replicons (possibly within a cluster) rapidly joined to each other. This is essentially what we observed, but the DNA was much larger than the 30 ~um (approx. 3 • 107 daltons) average rep- licon size of Chinese hamster cells [4]. While large replicons do occur in the size range o f the 1.4 • 108 dalton DNA, they are rather infrequent [4,8]. Thus, as noted above, our protocol may be selective for a unique DNA population. Experiments designed to elucidate replicon organization are in progress.

It is clear from the data in Fig. 3 that 2.75--3.0 h (e.g., one-third to one-half of S period) were required to chase the initially small DNA into bulk size DNA, a time not unlike that reported by others using different protocols [10]. If this is a general phenomenon for much of the cellular DNA, then the question arises as to the integration of DNA synthesized very late in the DNA synthetic period. If such late replicating DNA were to represent completion of relatively long replicons and/or replicons within a cluster, then a very rapid joining would allow integration into bulk DNA very soon after termination of DNA replica- tion. The other alternative, of course, would be that DNA synthesized in very late S is not integrated into bulk DNA and persists in an unintegrated form into G2.

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Although a good deal of work remains in further characterization, we feel that the system described above will allow examination of additional aspects of mammalian cell DNA replication which heretofore have remained elusive.

Acknowledgments

The authors wish to acknowledge the excellent technical assistance of John L. Hanners and Linda T. Ferzoco. This work was performed under the auspices of the U.S. Energy Research and Development Administration.

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