Cell, Vol.

12,1029-1043,

Asymmetric Replication

December

1977, Copyright

0 1977 by MIT

Okazaki Piece Synthesis during of Simian Virus 40 DNA in Vivo

Daniel Perlman Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02139 Joel A. Huberman Department of Medical Viral Oncology Roswell Park Memorial Institute Buffalo, New York 14263

Summary We have pulse-labeled simian virus 40 (SV40)infected monkey cells with 3H-thymidine (3HdThd) and have hybridized the viral Okazaki pieces (rapidly labeled short DNA chains found during DNA replication, < 250 nucleotides long) and SV40 “intermediate sized” DNA (longer nascent strands, up to full replicon size) to the separated strands of two SV40 DNA restriction fragments, one lying to either side of the origin of bidirectional DNA replication. As much as 5 fold more Okazaki piece DNA hybridized to one strand than to the other strand of each restriction fragment. The excess Okazaki piece DNA was in the strands oriented 3’ -+ 5’ away from the replication origin (the strands which are expected to be synthesized discontinuously). Neither the duration of the labeling period nor the temperature of the cells during labeling significantly altered this hybridization asymmetry. With respect to the hybridization of “intermediate sized” DNA, a reverse asymmetry was detected (1.7 fold more radioactivity in the strands oriented 5’ + 3’ away from the origin for a 1 min pulse label at 22°C). The effects on these hybridization asymmetries of preincubating the infected cells with FdUrd prior to pulse-labeling were also determined. We also measured the size of the Okazaki pieces using gel electrophoresis under denaturing conditons after releasing the pieces from the filter-bound DNA strands. The size distribution of the Okazaki piece DNA from each strand was the same (- 145 nucleotides, weight average; 200250 nucleotides, maximum size), indicating that the hybridization asymmetry resulted from a difference in the number rather than the size of the pieces in each strand. The simplest interpretation of our results is that SV40 DNA is synthesized semidiscontinuously: the strand with 3’ + 5’ orientation away from the origin is synthesized in short Okazaki pieces which are subsequently joine.d together, while the strand with 5’ -+ 3’ orientation away from the origin is synthesized continuously. Some models of two-strand discontinuous synthesis, however, cannot be ruled out.

Introduction An enzyme or enzyme complex which replicates double-stranded DNA must synthesize two chains having opposite chemical polarities. The DNA polymerases wh-ich have been purified from both procaryotic and eucaryotic cells are capable of elongating DNA in vitro only in the 5’ + 3’ direction. Experiments with both procaryotic and eucaryotic organisms suggest that as a replication fork proceeds, at least one strand is synthesized discontinuously by formation of short DNA chains which are subsequently joined to a longer nascent strand (reviewed by Okazaki et al., 1968; Edenberg and Huberman,l975). Such short chains are frequently called Okazaki pieces in honor of their discoverer (Okazaki et al., 1968). The phenomenon of discontinuous synthesis provides a mechanism by which the nascent strand which must be synthesized with overall 3’ + 5’ polarity can be produced by a polymerase synthesizing short chains only in the 5’ + 3’ direction. We shall refer to the nascent strand which is synthesized with overall 3’ + 5’ polarity as the “necessarily discontinuous” strand, and to the nascent strand which is synthesized with overall 5’ + 3’ polarity as the “potentially continuous” strand. There is controversy over whether replication of the potentially continous strand proceeds by continuous or discontinuous synthesis. Both procaryotic and eucaryotic experiments have yielded contradictory results. The case for discontinuous synthesis of both nascent strands has been supported by the finding that in some cases, ~50% of the radioactive precursor incorporated into DNA in very short pulses is incorporated into Okazaki pieces (Okazaki et al., 1968; Fareed and Salzman, 1972; Huberman and Horwitz, 1973; Gautschi and Clarkson, 1975; Kurosawa and Okazaki, 1975; Tseng and Goulian, 1975). Discontinuous synthesis has also been supported by the finding that in certain cases, a significant proportion of the viral Okazaki pieces purified from virus-infected cells is capable of self-annealing (Magnusson, 1973; Fareed, Khoury, and Salzman, 1973; Pigiet et al., 1973). On the other hand, the case for continuous synthesis of the “potentially continuous” strand has been supported by contrary findings. Some investigators have found that a maximum of 50% of pulse-labeled DNA can be detected in the Okazaki piece peak on alkaline sucrose gradients (Qasba, 1974; Francke and Hunter, 1974; Hershey and Taylor, 1974). In addition, some investigators have found that only a small proportion of purified viral Okazaki pieces are capable of self-annealing (Francke and Hunter, 1974; Francke and Vogt, 1975). The ambiguities inherent in such experi-

Cell 1030

ments and the difficulties in interpreting the results have been discussed elsewhere (Francke and Vogt, 1975; Edenberg and Huberman, 1975). A different method for determining whether Okazaki pieces come from one or both sides of the replication fork is to measure the hybridization of viral Okazaki pieces to the separated strands of viral DNA restriction fragments. This method allows direct measurement of the relative concentration of Okazaki pieces generated on each side of the replication fork. We believed it was important that such measurements be made on Okazaki pieces synthesized in vivo because of the possibility of artifacts during in vitro DNA synthesis. We selected the SV40 genome because, with the exception of initiation of replication (Tegtmeyer, 1972), all replication functions are specified by the host cell. SV40 should therefore be a useful probe for studying mammalian DNA replication. The origin and direction of replication have been determined for SV40; replication is bidirectional from an origin located at 0.67 on the standard restriction nuclease map of the SV40 genome (Danna and Nathans, 1972; see Figure 1). For the study described in this paper, we selected two restriction fragments of SV40 DNA, one lying to either side of the replication origin (Figure 1). The results of our study show that Okazaki pieces come predominantly, perhaps exclusively, from the “necessarily discontinuous” strand.

,

Restriction Frogments of SV40 DNA Used for Hybridizotion to Okozoki Fragments

Figure

1. Physical

Map of the SV40 Genome

The locations of the restriction enzyme cleavage sites relevant to this study (Hind ll+lll: Danna and Nathans, 1972; Eco RI: Morrow and Berg. 1972; Hpa II: Sharp, Sugden and Sambrook, 1973) and of the origin of replication (Danna and Nathans, 1972) are shown on this map. The polarity of the separated restriction fragment strands (“slow” and “fast” determined by mobility in agarose gel electrophoresis-see text) was determined by hybridization with E. coli RNA polymerase transcript (wavy line; see text). The orientation of “discontinuous” and “potentially continuous” nascent strands during DNA replication is also shown (zig-zag lines).

Results Preparation of Separated Strands of Restriction Fragments For use in these experiments, we selected the two restriction fragments of SV40 DNA illustrated in Figure 1. The Hind Il+lll-A fragment comprises about 0.225 of the SV40 genome and is located in the early region just to one side of the replication origin (Danna and Nathans, 1972), while the smaller of the two fragments generated by combined action of Hpa II and EGO RI, which we call the Hpa II-Eco RI-B fragment, is located in the late region just to the other side of the origin and comprises about 0.27 of the genome. We prepared these fragments, separated their strands by gel electrophoresis then fixed them to nitrocellulose filters, all as described in Experimental Procedures. Polarities of the Separated DNA Strands To determine the polarities of the separated DNA strands with respect to the entire SV40 DNA molecule, we were able to take advantage of the fact that E. coli RNA polymerase almost exclusively transcribes the “early” informational strand of

SV40 DNA (Westphal, 1970). This RNA is synthesized with polarity 5’ + 3’ in the counterclockwise map direction (Figure 1). Thus by hybridizing such RNA to the separated strands on filters, we could determine the polarities of the strands. 3H-UTP-labeled RNA was prepared by Don Coen in the laboratory of Alexander Rich using E. coli RNA polymerase and SV40 form I DNA. This RNA was hybridized in excess to half-filters containing the various DNA strands. The other half of each filter was used to measure the amount of DNA available on each filter for hybridization. For this purpose, we used uniformly labeled 3zP-SV40 DNA, which had been nicked and denatured before the hybridization, in a saturating amount. The results are shown in Table 1. Hybridization of the E. coli RNA polymerase transcript took place exclusively with the Hind Il+lll-A slow strand and the Hpa IIEco RI-B fast strand. These must therefore be part of the SV40 “early” DNA strand and have 3’ + 5’ counterclockwise polarity, as illustrated in Figure 1. The polarity assignments in Figure 1 imply that the fast strands of each restriction fragment are templates for “necessarily discontinuous” strand synthesis during bidirectional DNA replication,

Asymmetric 1031

SV40 DNA Synthesis

Table 1. Hybridization of RNA Transcribed from SV40 Form I DNA by E. coli RNA Polymerase to Separated Strands of SV40 Restriction Fragments W-RNA =P-SV40 DNA (Gross cpm)

DNA Filter E. coli Blank

Gross

Net

cm

cm

28

40

Hind Il+lll-A

Slow

172

288

248

Hind Il+lll-A

Fast

155

36

0 1

Hpa II-Eco

RI-B Slow

551

41

Hoa II-Eco

RI-B Fast

535

1366

1328

Two DNA filters of each restriction fragment strand were split in half. Half of each filter was assayed for relative DNA content by saturation hybridization with uniformly 32P-labeled SV40 DNA. The other halves were hybridized (in a single vial) with 30,000 cpm of aH-UTP-labeled RNA transcribed by E. coli RNA polymerase from SV40 form I DNA. Hybridization conditions are described in Experimental Procedures. Results are averages of two filters. Gross cpm = total cpm before subtraction of E. coli blank.

while the slow tially continuous”

strands are templates strand synthesis.

for “poten-

Purity of Separated Strands The data of Table 1 suggested that cross-contamination of slow strands with fast strands or fast strands with slow strands was slight (cl%). A separate experiment (data not shown) in which we hybridized radioactive separated strands to nonradioactive separated strands on filters confirmed that cross-contamination was slight (~5%). Thymine Asymmetry of the Separated Strands of Restriction Fragments If a significant difference existed in the thymine content of the complementary strands of the restriction fragments being used, then it would be necessary to normalize hybridization data obtained with 3H-dThd-labeled DNA. We therefore determined the relative thymine content of the DNA strands. We mixed 3H-dThdand uniformly 32POs labeled SV40 form I DNAs and then digested them to produce the restriction fragments used in this study. We then separated the DNA strands and determined the 3H to 32P isotope ratios for each strand, as well as for the original double-stranded restriction fragments. The 3H to 32P ratio was the same for both strands of each fragment within experimental error (data not shown), suggesting negligible thymine asymmetry. Preparation of Pulse-Labeled SV40 DNA We pulse-labeled infected BSC-1 cells between and 45 hr after infection. We placed petri plates

38 on

water baths at 37 or 22°C and then labeled them as described in Experimental Procedures. We terminated labeling by transferring the petri plates to an ice-water bath at OX, washing them thoroughly with ice-cold phosphate-buffered saline containing sodium azide and then lysing the cells with sodium dodecylsulfate (SDS) within 30 set of the end of the labeling. Alternatively, we lysed cells with NP40 to obtain nuclei and then lysed the nuclei with SDS. Keeping the cells at 0°C following the labeling until lysis should have prevented significant ligation of Okazaki pieces. We selectively extracted viral DNA from the SDS lysates by the procedure of Hirt (1967). and concentrated, denatured and sedimented the extracts through alkaline sucrose gradients. Typical sedimentation profiles are shown in Figures 2A, 2B, 3 and 5A. The profiles show the separation of pulse-labeled DNA into two components: a peak sedimenting at about 4s which has been shown in other laboratories to consist of Okazaki pieces (Fareed and Salzman, 1972; Salzman and Thoren, 1973; Francke and Hunter, 1974), and a broad peak with maximum sedimentation rate about 16s (determined using mature linear single-stranded SV40 DNA, which sediments at 16S, as a marker in similar gradients) which has been shown in other laboratories to contain nascent “intermediate sized” DNA strands (reviewed by Fareed and Salzman, 1972; Salzman and Thoren, 1973; Francke and Hunter, 1974; Edenberg and Huberman, 1975). By “intermediate sized” DNA we mean nascent DNA strands of variable length up to mature viral length which are the products either of continuous DNA synthesis or of the joining of Okazaki pieces. From these and other data (not shown), we deduced that labeling periods of 52 min at 22°C or 550 set at 37°C would permit the preparation of reasonably pure Okazaki pieces, minimally contaminated by “intermediate” strands. We modified this labeling procedure to obtain pulse-labeled DNA of higher specific activity. We transferred petri plates of infected cells to a water bath at 0°C and then incubated them in the presence of 3H-dThd at 0°C for 5 min. We then shifted the cells to a water bath at 22°C and labeled them with medium containing 3H-dThd at 22°C. We terminated labeling as above. Alkaline sucrose gradients of viral DNA from cells labeled only at 0°C for 5 min showed insignificant incorporation of 3H-dThd (data not shown). After 45 min at O”c, we could detect some synthesis, and this is described below (Figure 3D). The specific activity of viral DNA labeled at 22°C following preincubation at 0°C was 5-10 fold higher than that of DNA from nonpreincubated cells. When uninfected cells were labeled by either of the above procedures, we

Cell 1032

FRACTION Figure 2. Alkaline DNA Used in Filter

NUMBER

Sucrose Sedimentation Hybridizations

of Pulse-Labeled

SV40

(A) Cells were labeled for 1 min at 22”C, at 43 hr after infection. Sedimentation was for 20 hr at 14°C. (6) Cells were labeled for 45 set at 37°C. at 41 hr after infection. Sedimentation was for 19 hr at 14°C. (C) At 42 hr after infection, cells were incubated at 37°C with 10m5 M FdUrd for 30 min. The cells were then labeled with “H-dThd in otherwise identical medium at 21°C for 2 min. Sedimentation was for 18 hr at 12°C. The pooled gradient fractions (indicated by dashed line) were used in filter hybridizations (Table 2). Direction of sedimentation was from right to left.

could detect no radioactivity gradients of Hirt extracts.

in the alkaline

sucrose

Hybridization of Pulse-Labeled SV40 DNA to DNA Filters: Experiments Using a Saturating Internal Standard We wished to determine, as accurately as possible, the relative amount of pulse-labeled Okazaki piece or “intermediate sized” DNA which was comple-

mentary to each separated DNA strand. For this purpose, we used two methods of hybridization. The first method is described here. This method, saturation hybridization, consisted of mixing a saturating amount of nicked, denatured, uniformly 32P-labeled SV40 DNA with 3H-pulse-labeled DNA and co-hybridizing them to the filters. We placed both slow and fast strand filters, as well as an E. coli blank filter, in each hybridization solution. The resulting 3H to 32P ratios on the filters provided an indication of the relative amounts of 3H-DNA in the hybridization solution complementary to each separated strand. The 32P-DNA served as an internal normalization factor which corrected for variations in DNA content from filter to filter. The sources of 3H-pulse-labeled DNA used for all saturation hybridization experiments are listed in Table 2. Figure 2 shows the sedimentation profiles in alkaline sucrose gradients of some of these sources; Figure 2A shows the sedimentation profile of DNA from cells labeled at 22°C for 1 min, while Figure 28 shows the profile for cells labeled at 37°C for 45 sec. Figure 2C shows the sedimentation profile of DNA from cells labeled for 2 min at 21°C after a 30 min preincubation with FdUrd. As noted previously by Salzman and Thoren (1973) and by Laipis and Levine (1973), the DNA labeled after preincubation with FdUrd sedimented slowly. Comparison of Figure 2C with Figures 2A and 2B shows that although the DNA labeled after preincubation with FdUrd sedimented slowly, much of it sedimented more rapidly than normal Okazaki pieces. The results in Table 2 indicate that when pulselabeling is carried out without preincubation with FdUrd, there is 2-3 fold more Okazaki DNA hybridized to the fast than to the slow strand of each restriction fragment. The “intermediate” DNA, on the other hand, preferentially hybridizes to the slow strand of the Hpa II/Eco RI fragment, with a small bias. We obtained similar results for the Okazaki DNA regardless of labeling time or temperature over the range indicated, and regardless of whether the Hirt extract was prepared from whole cells or washed nuclei. These hybridization results suggested that a 2-3 fold excess of the “discontinuous” nascent strand (Figure 1) was present in the Okazaki peak from alkaline sucrose gradients, but that a significant amount of “potentially continuous” strand was also present. The fraction of input 3H-DNA hybridized represents approximately 20% of the theoretical maximum possible under nonsaturating hybridization conditions (see Discussion). The DNA labeled after treatment with FdUrd showed a much lower, perhaps insignificant, strand hybridization bias (Table 2). This symmetry was not unexpected, since all of the labeled DNA

Asymmetric

SV40 DNA Synthesis

1033

r 30

50

2c

00

10

50

E

E E

0%

c

k I.7

200

100

4,

i

I

iII V I ’I II

20

10

FRACTION Figure

3. Alkaline

Sucrose

0& 100

50

I I

I

30

I

40

NUMBER

Sedimentation

I

I

10

20

FRACTION

of Pulse-Labeled

SV40 DNA Used in Filter

I I

30

I

I.

4(

0

NUMBER

Hybridizations

All sedimentations were for 19 hr at 14°C. (A) Cells were pulse-labeled with 3H-dThd for 20 set at 22°C following preincubation at 0°C for 5 min in identical medium containing 5H-dThd, at 41 hr after infection. (8) At 46 hr after infection, cells were labeled for 1 min at 22°C as in (A). (C) At 42 hr after infection, cells were labeled for 45 set at 37°C without preincubation. Nuclei were prepared from these cells, as described in Experimental Procedures. (D) At 48 hr after infection, cells were transferred to 0°C and labeled for 45 min at that temperature.

sedimented slowly the effect of FdUrd

(Figure below.

2). We further

explore

Hybridization of Pulse-Labeled SV40 DNA to DNA Filters: Experiments Using Externally Standardized Filters under Nonsaturating Conditions In other experiments, the method of saturation hybridization described above proved to have some inherent problems (D. Perlman, unpublished results). The efficiency of 3H-DNA hybridization to the filters was reduced by the simultaneous hybridization between 3H and 32P-DNA going on in solution. In addition, it proved possible for 3H-DNA of the same strand as the DNA on the filter to be entrained to the filter by hybridization to 32P-DNA strands only part of whose length was base-paired to the filter DNA. We therefore tried a second method of hybridization to minimize these problems. We cut the DNA filters into three pieces of equal size and then assayed one filter third for DNA content by satura-

tion hybridization using an excess of uniformly labeled SV40 DNA. We then matched appropriate combinations of the remaining filter thirds for DNA content (to within 10%) and hybridized them with pulse-labeled DNA. To ensure that these hybridizations took place under nonsaturating conditions (that is, that the DNA on filters was in excess over hybridizable DNA in solution), we usually carried out two hybridizations in parallel with the two remaining thirds of the DNA filters. In these parallel hybridizations, we set the relative amount of 3HDNA in the two otherwise identical hybridization vials at approximately 2 to 1. We concluded that filter DNA was in sufficient excess when the filters from the vial with more 3H-DNA hybridized approximately 2 fold more 3H counts, and the ratio of counts hybridized to the fast strand versus the slow strand filters was the same for both vials. Under these conditions, the rate and integrated amount of 3H-DNA hybridized to each filter are proportional at all times to the concentration of complementary 3H-DNA in solution.

Cdl 1034

Table

2. Hybridization

of SH-Pulse-Labeled

Viral DNA Using

azP-Viral

DNA as Internal

Standard

Pulse-Label

Filter

DNA source

Time

Temperature

(SW

(“cl

Lysis

% of Input 3H-cpm Hybridized

f+s Net Total (3H/3zP cpm)

5H Fast/ 3H Slow (=P Normalized)

Hind II+lll-A

0

135

22

C

4

135/246

2.7

Hind

0 3

120

21

N

4

154/360

3.1

[: 0

45

37

N

5

141/410

2.7

Il+lll-A

Hind Il+lll-A Hpa II-Eco

RI-B

0

120

21

N

6

2601730

2.0

Hpa II-Eco

RI-B

0

45

37

N

5

1781775

2.2

Hind

0 (Figure

28)

45

37

C

3

Hpa II-Eco

Il+lll-A RI-B

0 (Figure

28)

45

37

C

5

2901427

2.6

Hpa II-Eco

RI-B

2.4

I (Figure

2A, pool

1)

60

22

C

4

2271753

0.66

,.

I (Figure

2A, pool 2)

60

22

C

3

193/819

0.68

II

0 (Figure

66

22

C

6

306/693

2.6

FdUrd Hind

64/I 08

Il+lll-A

Pool

II Hpa II-Eco

RI-B

2A. pool 3)

(Figure

2C)

1

120

21

C

0

2421550

0.76

Pool 2

120

21

C

7

4571180

1 .o

Pool 2 Pool 2 repeat

120 120

21 21

C C

6 4

3731067 385/ 1634

0.82 0.84

Conditions for hybridization are described in the text. Arrows Okazaki piece DNA; (I) “intermediate sized” DNA: (FdUrd) DNA 2C); (C) lysis of whole cells; (N) lysis of washed nuclei: (f+s net blank filter and isotope spillover; (‘H fasWH slow) ratio of net ratio.

connecting DNA sources indicate use of the same DNA preparation. (0) synthesized after 30 min of preincubation at 37°C with FdUrd (see Figure total) cpm hybridized to fast plus slow strand filters, corrected for E. coli 3H cpm hybridized to the two filters normalized by the 3zP fasWzP slow

The foregoing considerations apply mainly to the hybridization of 3H Okazaki piece DNA to filters. Solutions of “intermediate” strands must contain nearly equal molar quantities of DNA complementary to slow and fast strands. Thus the relative specific activity of the two strand types is being measured under all conditions. Provided that the filters are properly matched for DNA content, the degree of saturation of the filters should not affect the ratio of counts on the filters. Using this external standardization technique, we carried out the series of hybridizations summarized in Table 3. Figure 3 shows several representative alkaline sedimentation profiles of 3H-DNA preparations used in Table 3. The data for Okazaki sized” DNA in Table 3 piece and “intermediate show fast:slow strand ratios similar to, but somewhat more pronounced than, those of Table 2. For the reasons noted above, we consider the values in Table 3 to be more accurate than those in Table 2. The range of fast:slow strand ratios for Okazaki pieces in Table 3 is 2.5-4.0, with an average ratio of 3.0. As in Table 2, this ratio was not significantly affected by duration of labeling, temperature during labeling or method of cell lysis (within the ranges tested in Table 3). In addition, preincubation of cells with medium containing 3H-dThd at 0°C for 5 min preceding the labeling did not affect

the ratio. It is significant that in the shortest pulse (20 set), when the ratio of Okazaki DNA to intermediate DNA approached 1:l (Figure 3A), the hybridization asymmetry for Okazaki DNA remained essentially the same. The reverse strand hybridization ratio (slow:fast) exhibited by the “intermediate” DNAs tested in Table 3 was about 1.7:1, significantly lower in magnitude than the ratio for Okazaki pieces. In the series of experiments presented in Table 3, we also tested DNA isolated from cells preincubated with FdUrd for 15 min prior to labeling for 1 min at 37°C. Figure 4 shows the alkaline sedimentation profile of this DNA. The profile is different from that of DNA from cells labeled after a 30 min preincubation with FdUrd (Figure 2C): in addition to the large broad peak of slowly sedimenting DNA also seen in Figure 2C, about one third of the DNA in Figure 4 sedimented as rapidly as normal “intermediate” strands. When we hybridized DNA from the slowly sedimenting peak in Figure 4 to separated strands of restriction fragments, we found obvious strand asymmetry-in clear contrast to the near symmetry previously noted for the DNA from cells preincubated with FdUrd for 30 min (Table 2). The differences in sedimentation profile and strand asymmetry between the preparations of Figure 2C (Table 2) and Figure 4 (Table 3) suggest that ap-

Asymmetric 1035

Table

SV40 DNA Synthesis

3. Hybridization

of 3H-Pulse-Labeled

DNA using

Externally

Standardized

Filters

Pulse-Label

DNA Filters Hpa II-Eco

Hind

RI-B

Il+lll-A

Time

Temperature

DNA Source

(set)

(“c)

Lysis

0

60 ,/

22