Cell,

Vol. 12. 1021-1028,

December

1977, Copyright

0 1977 by MIT

In Vitro Polyoma DNA Synthesis: Short DNA Chains Tony Hunter, Bertold Francke The Tumor Virology Laboratory The Salk Institute P.O. Box 1809 San Diego, California 92112

and Lee Bacheler

Summary The kinetics of annealing of the separated strands of the polyoma DNA Hpa II restriction fragments 1 and 2 to an excess of purified short DNA chains isolated from in vitro pulse-labeled replicating polyoma DNA were determined. The results indicate that for each growing fork, the DNA strand which must grow discontinuously is represented about 4 times as frequently in the population of short DNA chains as the strand which could replicate continuously. In addition, the absolute concentration of short DNA chains in the two growing forks is approximately the same. The average size of the short DNA chains from the continuous strand was shown to be very similar to that of the short DNA chains from the discontinuous strand. We conclude that polyoma DNA replication in vitro proceeds by a predominantly semi-discontinuous mechanism. Introduction The DNAs of papovaviruses are replicated in the nuclei of their permissive host cells, and with the exception of a viral protein required for the initiation of replication (Tegtmeyer, 1972; Francke and Eckhart, 1973; Chou, Avila and Martin, 1974), all the steps in viral DNA synthesis are carried out by enzymes of the host cell. The use of in vitro DNA synthesis systems derived from papovavirus-infected fibroblasts has greatly increased our understanding of the detailed mechanism of DNA chain elongation in mammalian cells (Edenberg and Huberman, 1975). There is general agreement that viral DNA strand growth proceeds bidirectionally from a unique origin (Fareed, Garon and Salzman, 1972; Crawford, Syrett and Wilde, 1973) at least in part in a discontinuous fashion through the formation of RNA-primed short DNA chains approximately 150 bases in length (Fareed and Salzman, 1972; Magnusson et al., 1973, Francke and Hunter, 1974; Hunter and Francke, 1974b). These intermediates are subsequently joined to form progeny viral DNA strands through a series of steps involving the removal of the RNA primer, gap-filling by a DNA polymerase believed to be different from that involved in the synthesis of the short DNA chains and finally ligation (Magnusson et al., 1973; Salzman and Thoren, 1973; Laipis and Levine, 1973; Hunter and Francke, 1975).

Asymmetry

of

There is currently disagreement, however, whether this discontinuous synthesis mechanism operates on both sides of the two growing forks present in each viral replicative intermediate (totally discontinuous mode) or only on one side of each fork (semi-discontinuous mode). In the semidiscontinuous mode, the progeny strand, for which the chemical direction of synthesis (5’ to 3’) corresponds to the direction of movement of the growing fork, is elongated continuously. In the absence of any known eucaryotic DNA polymerases which elongate DNA chains in the 3’ to 5’ direction, it is believed that the other strand must be synthesized discontinuously. Two approaches have been used to distinguish between the two possible replication modes. The first involves kinetic analysis of the appearance of labeled precursors in long and short growing chains (Francke and Hunter, 1974). The second and potentially more reliable approach consists of examining to what extent the isolated short DNA chains can anneal to one another (self-annealing). We have already discussed at length the problems inherent in the interpretation of self-annealing data (Francke and Hunter, 1974). Early experiments with SV40 in vivo (Fareed, Khoury and Salzman, 1973) and polyoma in vitro (Magnusson, 1973), in which the short DNA chains showed the high self-annealing values (60%) expected for the totally discontinuous mode, can be criticized on a number of grounds. Two of these criticisms have been met by ensuring that the short DNA intermediates are purified free of potentially contaminating parental DNA through the use of density labeling and that virus stocks are completely nondefective. Even when short DNA chains meeting these criteria isolated from in vitro systems for polyoma DNA synthesis were used, both low (22%) (Francke and Hunter, 1974; Francke and Vogt, 1975) and high (60%) (Pigiet et al., 1973), self-annealing values were obtained. Both our kinetic analysis (Francke and Hunter, 1974) and our self-annealing data support the idea that polyoma DNA synthesis in vitro is predominantly semi-discontinuous. On the other hand, the results from Reichard’s group indicate that polyoma DNA synthesis may be totally discontinuous (Magnusson et al., 1973; Pigiet et al., 1973). It is possible that this discrepancy reflects a difference in the mechanism of viral DNA replication in the two systems which are prepared in different ways. We use a whole cell lysate of polyoma-infected BALB/3T3 cells incubated at 32°C in vitro, while Reichard and co-workers used washed nuclei from polyoma-infected Swiss/3T6 cells incubated at 25°C in vitro. It is clear that optimal synthesis of viral DNA in vitro, and in particular maturation of viral DNA, requires cytoplasmic factors (Francke and Hunter, 1975: Otto

Cell 1022

and Reichard, 1975; DePamphilis and Berg, 1975). Francke and Vogt (1975), however, reported that the self-annealing of short chains isolated from washed nuclei from polyoma-infected BALB/3T3 cells did not show any increase over that observed for short DNA chains from the lysate. Either the temperature of incubation or the cell type might account for the differences observed. Alternatively, the difference may be due to yet other shortcomings in the use of self-annealing to measure the concentration of short DNA chains complementary to the two parental DNA strands. For this reason, we decided to measure directly the relative amounts of short DNA chains from both sides of each of the two growing forks of polyoma replicative intermediates. To do this, we measured the rate of annealing of the separated strands of Hpa II fragments 1 and 2 (Figure 1) to a large excess of density-labeled, purified short DNA chains. The Hpa II restriction enzyme fragments 1 and 2 are of approximately equal size [27.3% and 21.4% of the polyoma DNA molecule, respectively (Griffin, Fried and Cowie, 1974)], are located on opposite sides of the origin of DNA replication, and are far enough removed from the origin to avoid problems with young continuously growing chains. In addition, they constitute a sufficiently large fraction of the genome to make the experiments technically possible. Our results indicate that there are approximately 4 times as many short DNA intermediates from the chain which must grow discontinuously (that is, the strand growing 3’ to 5’) as there are for the chain which theoretically can grow continuously. In addition, we find that there is essentially no difference in the size of the short DNA chains on the two sides of the growing fork. The significance of our results is discussed with regard to the two possible modes of DNA replication. Results Preparation of Short DNA Intermediates We decided to estimate the relative concentrations of short DNA chains from the two sides of both growing forks of polyoma replicative intermediates by measuring the rate of annealing in solution of the separated strands of Hpa II restriction fragments 1 and 2 to a large excess of short DNA chains. These chains were isolated after synthesis in the cell-free lysate of polyoma-infected fibroblasts that we have previously described (Hunter and Francke, 1974a). Although such an experimental design requires large quantities of purified short DNA chains, we believed that it was necessary to perform the experiment in this way, since our preparations of Hpa II restriction fragments 1 and

BIDIRECTIONAL ORIGIN

TERM-INUS Figure 1. Physical Map of Polarities for DNA Synthesis

Polyoma

DNA

Showing

the

Strand

The Hpa II fragments are ordered according to the map of Griffin et al. (1974). The strand polarities are derived from the polarity determined for the transcription of late mRNA (Kamen et al., 1974). By convention, the strand from which late mRNA is transcribed is designated L. In DNA replication, the growing forks move bidirectionally away from the unique origin of replication (Crawford et al., 1973). The continuous arrowed lines indicate the progeny strands which in theory can grow continuously; the broken arrowed lines indicate the strand which has to grow discontinuously.

2 contained small amounts of polyoma DNA sequences from other parts of the genome. This contamination would have complicated the interpretation of the results if we had used the separated strands of the restriction fragments in DNA excess hybridization to estimate the fraction of radioactivity in short DNA chains from the two sides of the growing fork. To carry out a kinetic analysis of annealing with each of the four separated strands of fragments 1 and 2 in a reasonable excess of purified short DNA chains, we needed approximately 500 ng of short DNA chains, since each restriction fragment only constitutes about one quarter of the viral genome. Due to uncertainties in the exact specific activity of the labeled precursors in the lysate and the small amount of DNA involved, it was difficult to determine the precise quantity of short DNA chains

Asymmetry 1023

of Short

Chains

in Polyoma

DNA Synthesis

prepared from the in vitro system by our standard procedure (Francke and Hunter, 1974; Francke and Vogt, 1975). Initial experiments indicated that we could prepare approximately 1 ng of density-labeled short DNA chains from every 9 cm dish used to make lysate. We therefore made a lysate from 500 9 cm dishes of polyoma-infected BALB/3T3 cells by our standard procedure (Hunter and Francke, 1974a), which we then labeled with (Y32P-dCTP in the presence of BrdUTP for 1.5 min. From this reaction mixture, we purified densitylabeled short DNA chains as previously described by selective Hirt extraction, neutral sucrose centrifugation, sizing by low salt neutral sucrose gradient centrifugation after denaturation of the replicative intermediates and finally equilibrium density gradient centrifugation in alkaline Cs,SO, (Francke and Hunter, 1974; Francke and Vogt, 1975). Our preparation differs from that previously described in that we included in our pool of DNA from the Hirt extraction, the free single-stranded DNA chains normally found at the top of the initial neutral sucrose gradient (Francke and Hunter, 1974). We thus ensured that we did not lose any polyoma-specific DNA by selecting viral replicative intermediates alone. This preparation of short DNA chains became 28% resistant to Sl nuclease digestion upon prolonged self-annealing, which is similar to values we have obtained previously (Francke and Hunter, 1974; Francke and Vogt, 1975). >95% of the short DNA chains became resistant to Sl nuclease digestion after annealing to a large excess of fragmented polyoma DNA, indicating that most of the short DNA chains were derived from viral rather than cellular DNA. Preparation of the Separated Strands of Hpa II Fragments 1 and 2 Purified 3H Hpa II restriction fragments 1 and 2 were separated into their complementary strands by hybridization with a large excess of cRNA as described in Experimental Procedures. The annealing properties of the separated strands used in the experiment in Figure 2 are listed in Table 1. The strands are designated E and L based on their transcription at early and late times after infection (Kamen et al ., 1974). The size of the separated strands was compared to that of the purified short DNA chains by alkaline sucrose gradient centrifugation. All the DNA fractions were found to be about the same size with the exception of the L strand of fragment 2, which contained somewhat shorter DNA chains. These short DNA chains did not hybridize well, as can be seen from their annealing values to cRNA and polyoma DNA (Table 1). For this reason, we have corrected the annealing values obtained with the L strand of fragment

s jj

2.5

r[ I? \

- 06

FRAGMENT

I

FRAGMENT

2

LATE 2.0

EARLY

0

3

6

9

I 12

I I!

” cot ” Figure 2. Annealing of Purified Short Polyoma DNA Chains to the Separated Strands of Hpa II Restriction Fragments 1 and 2 Hybridizations were carried out as described in Experimental Procedures. The graphs show values of the reciprocal of the fraction of the lH radioactivity in the separated strands of Hpa II fragments 1 and 2 remaining single-stranded, plotted against “Cot” on the abscissa. We have used the time of hybridization in hours corrected for the input concentrations of purified short DNA chains as determined by the input l*P radioactivity as our Cot value because we are unable to measure the absolute concentration of the short DNA chains. Least-squares best straight lines have been drawn through the points, and the slopes for these lines are given below. (m4) Vi radioactivity in L strand; (04) ‘H radioactivity in E strand. (A) Fragment 1: E-slope 0.112; L-slope 0.024; (Et) fragment 2: E-slope 0.026; L-slope 0.100.

2 by multiplying them by the reciprocal fraction of the radioactivity in the separated that annealed to excess polyoma DNA.

of the strand

Annealing of Separated Strands with Excess Short DNA Chains To measure the relative concentrations of short DNA chains from the two sides of both growing forks, hybridizations were performed in 0.5 M NaCl

Cell 1024

Table 1. Characterization Fragments 1 and 2

of the Separated

Fragment

1

Strands

of Hpa II

Fragment

Table

2. Annealing

of Short

DNA Chains % Sl Nuclease Resistance

2 Reaction

Time

of Hybridization

Zero

Time

Self-Annealing Annealing (24 hr)

(24 hr)

E

L

E

L

2.4

0.5

0.6

1.1

3.2

0.6

1.3

5.4

to cRNA

Annealing to Polyoma DNA (24 hr)

3.1 97.7

97.1 90.0

1.5 97.1

Short

DNA Chains

Alone

Short

DNA Chains

plus

20.4 Polyoma

(0.28/a)

DNA 100.0

65.1

Short DNA Chains plus Hpa II Fragment 1 E Strand

32.4

63.2

Short DNA Chains plus Hpa II Fragment 1 L Strand

24.6

Short DNA Chains plus Hpa II Fragment 2 E Strand

25.4

Short DNA Chains plus Hpa II Fragment 2 L Strand

26.6

Hybridizations were carried out as described in Experimental Procedures. The zero time samples were taken immediately after the reactions had been heated to 100°C for 5 min. The results are expressed as the percentage of the input radioactivity which became .%-resistant. The concentrations of the separated strands in the hybridizations were 4.1, 4.2, 5.6 and 4.6 nglml for the E and L strands of fragments 1 and 2. respectively. The concentration of cRNA was 4.7 pglml. The concentration of sonicated polyoma DNA was 2.3 wg/ml.

at 66°C as described in Experimental Procedures. The final concentration of the separated strand (3H) was in each case about 1 rig/ml and that of the short DNA chains (“*P) approximately 100 ng/ ml. Samples were taken at the times indicated, and the degree of annealing was assayed by Sl nuclease digestion. Table 2 shows the data for the final extent of annealing of the 32P radioactivity in short DNA chains in this experiment. We can use these values to give an indication of the actual excess of short DNA chains over separated strands in these reactions. For example, if the added strand were equal in amount to the short DNA chains complementary to that strand, then both strands of the fragment would have been hybridized to completion. If the ratio of the strands in the short DNA chains is 4:1, complete reaction of the E strand of fragment 1 would contribute an additional 16% hybridization over the value for self-annealing. Complete reaction of the L strand of fragment 2 would contribute 13% additional hybridization. The small size of the increases in the level of annealing in the presence of the separated strands of fragments 1 and 2 suggests that the reactions contained a reasonable excess of short DNA chains over the separated strands (Table 2). The majority of the 32P radioactivity annealed presumably represents self-annealing of the short DNA chains. The values for the annealing of the 3H radioactivity in the separated strands are plotted as the reciprocal of the fraction remaining against Cot (Figure 2). In such a plot, the slope of the line is directly proportional to the rate of annealing and hence to the concentration of the DNA sequences annealed to the labeled DNA (Wetmur and Davidson, 1968). In Figure 2A, it can be seen that the E

The short DNA chain preparation used in the experiment in Figure 2 was annealed either alone or in the presence of an excess of fragmented polyoma DNA at a concentration of 1.5 x 10’ 32P cpmlml. The reactions with the separated strands (Figure 2) were carried out at a concentration of 1 x 10’ 3zP cpm/ml. The values of resistance to Sl nuclease achieved after 11 hr of hybridization are listed for each reaction.

strand of fragment 1 hybridizes approximately 4 times faster with excess short DNA chains than does the L strand of fragment 1. In contrast, the annealing of the L strand of fragment 2 is about 4 times faster than the E strand of fragment 2 (Figure 28). From the known polarities of the E and L strands of polyoma (Kamen et al., 1974), we can predict that the synthesis of the complements of the E strand of fragment 1 and the L strand of fragment 2 must be in the 3’ to 5’ direction (see Figure 1). Complements to the L strand of fragment 1 and the E strand of fragment 2, on the other hand, are synthesized in the 5’ to 3’ direction. Thus the strand which has to grow discontinuously is represented approximately 4 times more frequently in the population of short DNA chains than that which grows in the 5’ to 3’ direction. It is worth noting that this ratio is numerically consistent with the self-annealing values (22-40%) observed for the short DNA chains isolated from our in vitro system (Francke and Hunter, 1974; Francke and Vogt, 1975). Even though both types of data suggest a predominantly semi-discontinuous mode of synthesis, however, it should be stressed that there is a significant and measurable concentration of short DNA chains from the continuous strand. Since the specific activities of the separated strands of Hpa II fragments 1 and 2 are identical, we can also calculate from the data of Figure 2 that the absolute concentrations of short DNA chains from the discontinuous strand in each growing fork are approximately the same. This is also true for the lower levels of short DNA chains coming from the continuous strand.

Asymmetry

of Short

Chains

in Polyoma

DNA Synthesis

1025

Sizing of Short DNA Complements to Fragment 1 The results of the preceding experiment support the idea that the synthesis of polyoma DNA in vitro is predominantly semi-discontinuous. It is possible, however, to account for the paucity of short DNA chains coming from the potentially continuous strand in a totally discontinuous mode if the rate of joining of these chains is faster than on the discontinuous strand (Kainuma-Kuroda and Okazaki, 1975; Kurosawa and Okazaki, 1975). This explanation predicts that the short DNA chains corresponding to the continuous strand should on average be shorter than those from the discontinuous strand. To test this prediction, we have estimated the size of the short DNA chains complementary to the two sides of the growing fork. Purified short DNA chains were fractionated on an alkaline sucrose gradient (Figure 3A), and the concentrations of sequences complementary to the separated strands of Hpa II fragment 1 from each gradient fraction were measured. For this experiment, Hpa II fragment 1 was labeled to a higher specific activity in vitro (2 x 10’ cpm/pg) than we could obtain by labeling polyoma DNA in vivo (3 x lo6 cpmlpg) to ensure that an excess of short DNA chains could be achieved for each gradient fraction. We hybridized an equal aliquot of each gradient fraction to the E and L strands of fragment 1 for 20 hr at 68°C in 0.5 M NaCI. At this time of hybridization and concentration of short DNA chains, the annealing values of the separated strands are proportional to the concentration of complementary sequences. Figure 3B shows the distribution of short DNA chain complements to the E and L strands of fragment 1. Consistent with our previous experiment, the concentration of complements to the E strand of fragment 1 is greater than the concentration complementary to the L strand. Within the resolution of our experiment, the complements to the L strand of fragment 1, corresponding to the continuous strand, are the same size as the complements to the E strand from the discontinuous strand.

500 400

I

I

I

25

15

&KIN

I

2 2 m

I

30 NUMBER

35 +

100 -@ ii go-

I

I I1 I I I1 I 1lImmnnrmxlulxx POOL NUMBER

Figure 3. Sizing Fragment 1

of the Complements

I

I

to the E and

L Strands

of

Purified short DNA chains (see Experimental Procedures) were further fractionated on an alkaline sucrose gradient (520% linear sucrose gradient in 0.75 M NaCI, 0.25 M NaOH, 0.01 M EDTA) in an SW41 rotor for 38.5 hr at 39,000 rpm at 4°C. Sedimentation was from right to left. (A) The distribution of 32P radioactivity in short DNA chains in the relevant region of the alkaline sucrose gradient (A-A). Also indicated are the sizes (in bases) (-) of the single-stranded short DNA chains estimated from the marker polyoma DNA Hpa II restriction fragments 7 and 8 run in a parallel gradient. (6) Adjacent fractions from the gradient in (A) were pooled and hybridized to the E and L strands of fragment 1 as described in the text and Experimental Procedures. The fraction of Y-f radioactivity in the separated strands which became Sl nucleaseresistant is plotted for each pooled gradient fraction. (O-O) E strand; (m--m) L strand.

Discussion The experiments described in this paper were undertaken to provide a direct measure of the steady state concentration of short DNA chains on either side of the growing forks of polyoma replicative intermediates. The kinetics of annealing of each of the separated strands of Hpa II fragments 1 and 2 with an excess of short DNA chains indicate that for each growing fork, the DNA strand which must grow discontinuously (that is, the complements to the E strand of fragment 1 and the L strand of fragment 2) is represented about 4 times as fre-

quently in the steady state population of short DNA chains as the DNA strand which could replicate continuously (see Figures 1 and 2). Taken at face value, these results support the idea that polyoma DNA replication occurs via a semi-discontinuous mechanism. Is it possible to account for our results if the mode of synthesis is discontinuous on both strands (totally discontinuous mode)? An appealing explanation has recently been put forward to account for the paucity of short DNA chains from the potentially continuous strand during replication of phage

Cell 1026

P2 DNA (Kainuma-Kuroda and Okazaki, 1975; Kurosawa and Okazaki, 1975). It is proposed that the rate of gap filling and joining is considerably faster on the continuous strand than on the discontinuous strand due to the fact that the gap-filling process can start before the synthesis of a short DNA chain on this strand is complete. On the discontinuous strand, however, the gap-filling process cannot begin until the synthesis of the short DNA chain is complete. If the units of discontinuous synthesis are the same size on both sides of the growing fork, then this explanation predicts that there should be a lower concentration of sequences in short DNA chains coming from the continuous strand, and that these chains should, on average, be shorter than those from the discontinuous strand. Since for our system the concentration of sequences in the population of short DNA chains derived from the continuous strand is one quarter that from the discontinuous strand, one would predict that the short DNA chains from the continuous strand should, on average, be one quarter the length of those from the discontinuous strand, provided there are equal numbers of short DNA chains on each side of the growing fork. The short DNA chains complementary to the L strand of fragment 1, however, appear to be approximately the same size as those complementary to the E strand of fragment 1 (see Figure 3). If the mechanism of DNA synthesis is totally discontinuous, then because of the asymmetrical strandedness, the short DNA chains on the continuous strand can only be the same size as those on the discontinuous strand if they have a much shorter average lifetime. Since the synthesis time for a short DNA chain is probably considerably less than its lifetime (Francke and Hunter, 1974), this possibility cannot be ruled out. It appears improbable, however, that polyoma DNA replication proceeds by a totally discontinuous mechanism. Another interpretation of our results is that the mechanism of DNA synthesis is not exclusively of one type or the other-that is, synthesis on the continuous strand proceeds discontinuously only in part. This would mean that reinitiation on the continuous strand is optional. The parameters governing the length of short DNA chains in eucaryotic cells are not known, but the fact that they are ~200 bases long while the short DNA chains found in procaryotes are much longer (Edenberg and Huberman, 1975) suggests that chromosomal structure may be important. The possibility that structural rather than sequence signals are involved in the initiation of short DNA chains is supported by the random RNA/DNA link at the 5’ end of short chains (Magnusson et al., 1973; Hunter and Francke, 1974b; Pigiet, Eliasson and Reichard, 1974). Eucaryotic DNA is packaged in

200 base pair units known as nucleosomes (Kornberg, 1974), and an attractive idea is that the unfolding of a single nucleosome is the signal for the initiation of a short DNA chain, thereby generating 200 base long chains on the discontinuous strand. On the continuous strand, there may be the option of either initiating a short DNA chain de novo at such a structural signal or, alternatively, of continuing to extend the progeny strand. The factors governing this optional reinitiation might include, among others, availability of the appropriate RNA and DNA polymerases and the speed of unfolding of the next nucleosome. If, as we believe, the mechanism of synthesis of polyoma DNA is semi-discontinuous, how does one account for the short DNA chains coming from the continuous strand? Since Hpa II fragments 1 and 2 are rather near the terminus of replication, it is possible that the existence of these chains is accounted for by overlapping termination. It is known that for SV40 there may be considerable variability in the exact site of termination from molecule to molecule (Lai and Nathans, 1975; Chen, Birkenmeier and Salzman, 1976). Additionally, a unidirectional origin of replication in a small fraction of replicating molecules, such as that described by Robberson et al. (1975), might give rise to short DNA chains from the L strand of fragment 1 and the E strand of fragment 2. We believe, however, that these explanations are not sufficient to account for all these short DNA chains. In particular, the agreement between the actual degree of self-annealing (-30%) of the short DNA chains and the self-annealing value predicted from the relative concentrations of short DNA chains on the two sides of the growing forks in Hpa II fragments 1 and 2 (-40%) indicates that the short DNA chains on the continuous strand are probably present throughout the entire genome. Such a uniform distribution of short DNA chains on the continuous strand might be found if the short chains were not intermediates in the synthesis of that strand per se, but arose from repair processes correcting errors in the newly synthesized progeny strand. This type of short chain could be distinguished from true intermediates by the absence of an RNA primer. Although it is clear that most of the short DNA chains in polyoma replicative intermediates are RNA-primed (Pigiet, Eliasson and Reichard, 1974), it is not known whether this is the case for the minority of chains coming from the continuous strand. Taking all these possibilities into consideration, we believe that it is most probable that polyoma DNA is replicated by a semi-discontinuous mechanism. In experiments similar in design to the ones reported here, Reichard’s group, using purified nuclei, has also found a preferential synthesis of

Asymmetry 1027

of Short

Chains

in Polyoma

DNA Synthesis

short DNA chains coming from the discontinuous strand (P. Reichard, personal communication; J. Flory, personal communication). The difference between the amounts of fragments from the two sides of the growing fork is, however, smaller than in our case, but is in line with the higher degree of self-annealing in their system. They also find that the two classes of fragments differ little in size. In general, the results obtained in the two laboratories now appear to agree quite well, and the remaining quantitative differences may be explained by the different experimental systems. Despite the advantages of using in vitro systems for analysis of the detailed mechanism of DNA synthesis, one is aware that the DNA synthetic machinery may be subtly altered -for instance, by the introduction of nonphysiological rate-limiting steps-during the preparation of the system. It is therefore reassuring that Perlman and Huberman (1977) have recently shown that SV40 replicative intermediates pulse-labeled in vivo have at least 3 times as many short DNA chains on the discontinuous strand as on the continuous strand. In addition, they showed that the short chains from the two sides of the growing fork were approximately the same size. It is striking that the relative levels of pulse-labeled short chains on either side of the growing forks of SV40 replicative intermediates are about the same as we find for the steady state levels in polyoma replicative intermediates in vitro. This similarity implies that the lifetimes of the short chains on the two sides of the growing fork are nearly the same. Penman and Huberman (1977) were also able to show that pulse-labeled DNA chains of intermediate length had an opposite bias in their strandedness, with approximately twice as much radioactivity coming from the continuous strand as from the discontinuous strand. Their results suggest that a predominantly semi-discontinuous mechanism is also operating in vivo. A definitive answer, however, to the question of whether the mechanism of DNA synthesis in vivo is totally, semi- or partially discontinuous requires either conditional mutants in the enzymes of DNA synthesis or else specific inhibitors (such as monospecific antisera) of the enzymes involved Experlmenial

Procedures

Cells, Vlrur and lnkctlon Condltlons (Hunter and Francke, 1974a) BALB/3T3 cells were grown and infected with plaque-purified polyoma ts.1260 virus at an moi = 20 PFU per cell as described. The Hpa II restriction fragment pattern was checked for each virus stock to ensure that it was completely nondefective (Francke and Vogt, 1975). In Vltro DNA Syntheslr (Hunter and Francke, 1974a; Francke and Hunter, 1975) For the experiment shown in Figure 2, the lysate from 500 9 cm plates was labeled half a minute after the start of the incubation

at 32°C with ~Y-~~P-~CTP (97 Cilmmole, NEN) at 20 @/ml for 1.5 min in the presence of 250 PM BrdUTP (Terramarine, La Jolla, California) with the omission of TTP. The reaction was carried out in 24 3 ml aliquots to avoid problems with mixing and temperature equilibration. For the experiment in Figure 3, the lysate from 100 9 cm plates was labeled 1.5 min after the start of the incubation at 324: with a-=P-dGTP (135 Ci/mmole, NEN) at 20 &i/ml for 2 min in the presence of 250 @A BrdUTP with the omission of TTP. The reaction was carried out in five 2 ml aliquots. Purlfkatlon of Short DNA Chalns (Francke and Hunter, 1974; Francke and Vogt, 1975) Density-labeled short chains were purified essentially as described, except that the phenol extraction preceded the initial sucrose gradient, and that the free single-stranded DNA chains at the top of the neutral sucrose gradient were pooled together with the viral replicative intermediates for subsequent steps. Preparation of Hpa II Restrktlon Fragments (Vogt, Bacheler and Bolce, 1976) Polyoma form I DNA labeled in vivo with ‘H-dThd to a specific activity of 2.8 x lp cpm/pg was digested with Hpa II restriction enzyme as described. The digest was applied to a 13 x 1 cm 2.% acrylamide, 0.1% bisacrylamide, 0.7% agarose gel, and the individual fragments were collected by continuous electroelution at 50 V. The fragments were concentrated by phenol extraction and precipitation with ethanol in the presence of yeast RNA. Fragments 1 and 2 were shown to be ~1% cross-contaminated with intact fragments by rerunning on analytical 3.5% acrylamide gels. For the experiment in Figure 3. Hpa II fragment 1 isolated as described above from SH-polyoma form I DNA (3 x IO6 cpm/pg) was labeled by nick translation in a reaction mixture containing 0.05 M potassium phosphate buffer (pH 7.5), 0.005 M MgCI,, 5 pg/ml DNA, 0.2 pglml DNAase I (Worthington), 30 FM SH-dTTP (47 Ci/mmol. NEN), 60 pM dCTP, 60 PM dGTP, 80 PM dATP and 30 units per ml DNA polymerase I (a gift from Dr. lnder Verma) as previously described (Bacheler. 1976). The fragment labeled to a specific activity of 2 x lO’cpm/pg was separated into its complementary strands as described below. Separatlon of Stranda of Fragmenta 1 and 2 The preparations of fragments 1 and 2 were sonicated 4 times for 30 set at a power setting of 4 with the microtip (6 watt output) of a Elranson Sonifier at a concentration of 2 fig/ml in 10 mM TrisCl (pH 7.5). 1 mM EDTA. Complementary RNA (cRNA) to polyoma DNA was prepared as described (Kamen et al., 1974) using form I polyoma DNA and E. coli RNA polymerase at 0.45 M KCI. Complementary sequences in the cRNA were removed after self-annealing by passage over Whatman CFll cellulose (Franklin, 1968). The sonicated, separated strands of the Hpa II restriction fragments were annealed to the asymmetric cRNA in RNA excess, and the strands were separated as described by Kamen et al. (1974), except that a column of hydroxylapatite was used. After a second cycle of self-annealing and hydroxylapatite fractionation, the separated strands were dialyzed against 10 mM Tris-Cl (pH 7.5), 1 mM EDTA and ethanol-precipitated in the presence of calf thymus DNA. The properties of the separated strands are shown in Table 1. The separated strands of fragments 1 and 2 are designated E and L according to the convention described in Figure 1. Hybrldlxatlons Hybridizations were carried out in 0.5 M NaCl, 0.01 M TES (pH 7.0) 0.001 M EDTA and 0.1% SDS in siliconized glass tubes. Samples were heated to 100°C for 5 min before being quenched in ice for removal of zero time samples. For the experiment described in Figures 2 and 3, the reaction was carried Out at 68°C with 250 ~1 samples being removed at appropriate times and diluted with 250 1.11Sl digestion buffer containing 0.1 M sodium

Cdl 1026

acetate (pH 4.5), 0.005 M ZnSO1, 50 wg/ml denatured calf thymus DNA and 5 pg/ml native calf thymus DNA. When necessary, Sl nuclease (Miles) was added to a final concentration of 500 units per ml and digestion was for 2 hr at 45°C. Resistant nucleic acids were precipitated in the presence of 50 pg yeast RNA and filtered onto Millipore HA filters. For other hybridizations, suitably sized samples were diluted at least 20 fold with 0.25 M NaCI, 0.05 M sodium acetate (pH 4.5) 0.0025 M ZnSO,, 25 wg/ml denatured calf thymus DNA, 2.5 pglml native calf thymus DNA and digested with Sl nuclease as above. The values shown in Figures 2 and 3 have been corrected for counter background and 3zP overlap, but not for the zero time Sl nuclease resistance of the separated strands of the Hpa II restriction fragments.

We thank Dr. Marguerite Vogt for her generous gift of purified Hpa II restriction enzyme fragments 1 and 2, Deana Fowler for expert technical assistance and Dr. Joel Huberman for communicating his results prior to publication. We are most grateful to the entire Tumor Virology Laboratory for their patience in listening to and critizing the arguments and experiments presented in this paper. In addition, we would like to acknowledge the stimulus of Dr. Peter Reichard in the design of these experiments. This research was supported by grants from the NIH to T. H. and 6. F. L. T. B. held an American Cancer Society postdoctoral fellowship, followed by a fellowship from the California Division of the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 U.S.C. Section 1734 solely to indicate this fact. July 5, 1977;

revised

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In vitro polyoma DNA synthesis: asymmetry of short DNA chains.

Cell, Vol. 12. 1021-1028, December 1977, Copyright 0 1977 by MIT In Vitro Polyoma DNA Synthesis: Short DNA Chains Tony Hunter, Bertold Francke Th...
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