DNA elongation rates and growing point distributions of wild-type phage T4 and a DNA-delay amber mutant

J. Mol.

Bid.

(1976)

106, 963-981

DNA Elongation Rates and Growing Point Distributions Wild-type Phage T4 and a DNA-delay Amber Mutant

of

DAVID MCCARTHY, CHARLES SKINNER HARRIS BERNSTEIN AND CAROL BERNSTEIN

.Molecular Biology Program, Microbiology Department College of Medicine, University of Arizona Tucson, Ark. 85724, U.S.A. (Received20 April 1976, and in revised form 25 June 1976) The rates of DNA elongation by wild-type phage T4 and a gene 52 DNA-delay awa mutant were estimated by pulse-labeling infected cells with tritiated thymidine and visualizing the gently extracted DNA by autoradiography. The estimated rate of chain elongation of wild-type DNA was 749 nucleotides/second early in synthesis and 516 to 581 nucleotides/second at 8 later time. The rate of DNA elongation by the am mutant was measured to be 693, 758 and 829 nucleotides/second during successive stages of synthesis, indicating that elongation was not slower than in wild-type. The kinetics of DNA increase after infection of host cells by wild-type phage T4 or by the gene 52 DNA-delay am mutant was followed using [methyL3H]thymidine uptake into acid-insoluble material. It was found that DNA increase in both wild-type and am infections could be represented as exponential during early times and linear during late times of DNA synthesis. From the rates of DNA increase and the rates of DNA elongation we were able t,o estimate the number of growing points per chromosome equivalent of templato DNA during the exponential and linear phases. Our estimates for wild-type phage were 0.55 and 0.7 1 to 0.80 growing points per chromosome equivalent of template DNA in the exponential and linear phases, respectively. For the urn mutant we found 0.14 and 0.12 to O-13 growing points per chromosome equivalent of template DNA during the exponential and linear phases, respectively. The apparent lower incidence of growing points in the am mutant infections suggests that the mutant may be defective in the initiation of growing points.

1. Introduction An autoradiographic procedure for visualizing intracellular phage T4 DNA was recently developed (Bernstein & Bernstein, 1973). We report here an application of this procedure to a study of the replicative fork. The method allows an estimation of the rates of movement of the growing point at intervals during infection and gives some insight into the relationships of the forks to each other as well as to the overall concatemeric DNA complex. Combining the information on elongation rates with measurements of total DNA increase, we can estimate the mean number of growing points per chromosome equivalent of template DNA. Phage T4 am mutants defective in the four genes 39, 52, 58-61 and 60 (the so-called DNA-delay genes) when grown on a restrictive host characteristically show a very slow initial rate of DNA increase at 37°C (Yegian et al., 1971) and little or no synthesis at 25°C (Mufti $ Bernstein, lx3

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1974). Autoradiographic and kinetic experiments with a representative DNA-delay mutant indicate that although rates of growing point movement are approximately the same as in wild-type infections, the number of growing points per unit of template DNA is much reduced. The implications of these results for understanding the role of the DNA-delay genes in replication are considered. am

2. Materials

and Methods

(8) Strains and media E8cherichia coZi strains S/6/5, CR63 and B3R were used. S/6/5 and CR63 are the standard restrictive and permissive hosts, respectively, for T4 am mutants (Epstein et al., 1963). E. coli B3R is a derivative we have selected from E. coli B3. It differs from the parent strain in that it does not form filaments and does not have reduced viability when incubated in media containing [methyL3H]thymidine at high spec. act. It retains the parental characteristics of being a low thymidine-requiring strain and being restrictive for am mutants. T4D+ (wild-type), amNG576 (gene 52, DNA-delay), amC21 (gene 33, maturation defective) and amNG661 (gene 55, maturation defective) were the phage strains used in this study. In experiments in which labeling was done at 2 specific activities of [methyl3H]thymidine the phage mutant t&3 (thymidylate synthetase deficient) was used to allow accurate adjustment of the level of label incorporated and to insure high levels of incorporation of exogenous thymidine. All cultures used in labeling phage DNA were incubated in M9 medium (Adams, 1959) supplemented with FeC1,*6H20 (2.7 rg/ml). Additional supplements are as described in the following section. H-broth (Hershey) was used for growth and suspension of the indicator bacteria used in plating phage. EHA bottom and top agar were used for plaque assays or for plating bacteria (Steinberg & Edgar, 1962). (b)

Growth conditions

and

ZabeZing of the DNA

For experiments measuring the incorporation of [methyZ-3H]thymidine into acidinsoluble material, E. coli S/6/5 from overnight cultures was grown to 4 x 10s cells/ml at 37°C in M9 medium supplemented with 10 pg thymidine/ml. Phage were then added at a multiplicity of infection of about 11. Since infections were carried out without prior addition of a metabolic inhibitor (e.g. KCN), superinfection inhibition and delayed lysis would not be prevented. At 1 min after infection, a solution of [methyZ-3H]thymidine Boston, Mass.) and 2’-deoxyadenosine (40 to 60 Ci/mmol; New England Nuclear Corp., was added at final concns of 8.9 rg/ml (0.27 Ci/mmol) [methyZ-3H]thymidine, and 219 pg 2’-deoxyadenosine/ml. At various times Oal-ml samples of the culture were diluted into equal volumes of 1 M-KCN to inhibit further metabolism. Portions (0.1 ml) of the diluted samples were placed on Whatman 3MM filter papers (2.3 cm) and dried at 85°C for 45 min. After drying, the filters were washed 3 times in 5% (w/v) trichloroacetic acid for 10 min, once in 95% (v/v) ethanol for 5 min and once in acetone for 5 min. All washes were done at 5°C. When dry, the washed filters were placed in glass scintillation vials containing Arlington Heights, Ill.). Samples 6 ml Spectrafluor/PPO/POPOP (Amersham Searle, were counted in a Nuclear Chicago mark I liquid scintillation counting system to a 2% error. Data were plotted after subtracting the background counts. For pulse experiments performed at a single spec. act. of [methyZ-3H]thymidine, infection was as described above except that a culture of S/6/5 was grown in M9 medium supplemented with 250 pg 2’-deoxyadenosine/ml. At various times portions of the infected culture were diluted into equal volumes of prewarmed MS medium containing [methyl3H]thymidine (60 Ci/mmol, 8 rg/ml). B3R was For pulse experiments performed at 2 spec. act. of [methyZ-3H]thymidine, prelabeled by growing for about 7 generations at 30°C to 1 x 10s cells/ml in M9 medium supplemented with 0.5 mg Difco vitamin-free Casamino acids/ml and 4 pg [methyl3H]thymidine/ml (6.7 Ci/mmol; New England Nuclear Corp., Boston, Mass.). Bacterial

DNA

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IN PHAGE

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965

DNA was prelabeled to prevent dilution of the [methyZ-3H]thymidine pool upon breakdown of the bacterial DNA. The cells were centrifuged in the cold and resuspended at 4 x lOs/ml in medium of the same composition but also containing phage td8 at a multiplicity of infection of 10. At 25 min after infection, during the linear phase of phage DNA synthesis, t#he infected cells were centrifuged in the cold and resuspended in prewarmed medium of the same composition but now supplemented with 4 pg [naethyZ-3H]thymidine/ml (52 Ci/mmol) to allow DNA labeling at a higher spec. act. At 5 min after adding the high spec. act. label, a 0.1.ml sample of the infected cells was removed for lysis and autoradiography. (c) Lysis and autoradiography procedures Infected cells were lysed and their DNA was dialysed and deposited on Millipore filters as described by Bernstein & Bernstein (1973). Mature chromosomes were extracted and dialysed as described by Bernstein (1970). Autoradiography was carried out as described by Bernstein (1970), except that exposures varied from 30 days for the single spec. act,. experiment to 90 days for the double spec. act. experiment, and Kodak D19 developer was used. For each kind of DNA, 4 to 6 Millipore filters were examined by autoradiography. (d) Length measurements and grain counting The procedure used for measuring the lengths of grain tracks was to mount the autoradiographs on microscope slides, and then to project the image obtained through the microscope by television camera on to a television screen. The images of the grain tracks were measured directly on the screen. As a length standard the image of a stage micrometer was similarly projected and the length of the 50 pm calibration measured on t,he screen. The final magnification of the image was 2270 x . Grain counting was carried out on photographic enlargements of the autoradiographs as described previously (Bernstein, 1970).

3. Results (a) Identification

of replicative

fork8

An initial autoradiographic experiment was performed using two levels of labeling to identify replicative forks of concatemeric replicating DNA. As described in Materials and Methods, the infected cells were first grown with [methyl-3H]thymidine at 6.7 Ci/mmol. After 25 minutes growth at 30°C the cells were transferred to medium containing labeled thymidine at the higher specific activity of 52 Ci/mmol. The infection was stopped after five minutes of high specific activity labeling. The cells were then lysed and the DNA spread on Millipore membranes for autoradiographic visualization. It is expected that in DNA undergoing semiconservative replication the newly replicated DNA would have a 4.4-fold ((52+6.7)/(6*7+6.7)) higher specific activity than the previously labeled DNA. Figure 1 shows two densely labeled replicative forks as part of a lightly labeled DNA structure which appears to be a single concatemer about 2830 pm long (portions of it extending beyond the photograph). In fork A the grain densities of the two densely labeled branches were 3.7 and 3*6-fold higher, respectively, than nearby portions of lightly labeled DNA, and in fork B the grain densities of the branches were 4.0 and 3*7-fold higher. These ratios are close to the ratio of 4.4 predicted solely on the basis of semiconservative replication. The somewhat lower ratios obtained are expected because coincidence of grains in the more densely labeled regions leads to undercounting. The lengths of the labeled branches in fork A were 64.5 pm and 64.0 pm, and in fork B were 64.0 pm and 60.0 pm, indicating that the rates of replication were the same in both forks and in both arms of each fork. The rates of elongation calculated from the two branches of fork A and the two of fork B were 12.9, 12.8, 12.8 and 12.0 pm per minute, respectively. This

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FIG. 1. Phage T4 replicative complex phage were grown in low spec. act. [ntethyL3H]thymidine (6.7 Ci/mmol) from 0 to 26 min and high spec. act. label (62 Ci/mmol) from 26 to 30 min. Intracellular growth was stopped at 30 min. Cells were lysed and the DNA allowed to adhere to Millipore membranes. (A) Replicative loop elongating in only one direction. (B) Replicative fork. (C) Six strands converging at a small circle of DNA. (D), (E) and (F) Densely labeled segments. Magnification 380x.

DNA

REPLICATION

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corresponds to rates of 1034, 1026, 1026 and 962 nucleotides per second calculated by using the length standard described in section (e) below. Fork A is part of a replicative loop in which the opposite fork is not densely labeled. This configuration is most simply interpreted as a unidirectional replicative loop. There is a small, lightly labeled ring (designated C in Fig. 1) at which six of the strands forming the complex appear to converge. It is possible that this reflects a remnant of an originally compactly coiled ring structure (Bernstein & Bernstein, 1974). Further examples of rings are shown in Figure 2 (i) and (j). Three other densely labeled segments (Fig. 1, D, E and F with D extending beyond the margin of the photograph) have grain densities 3.6, 3.3 and 3.2-fold higher than the lightly labeled DNA, and lengths of about 61 pm, 71 pm and 76 pm, respectively. These characteristics suggestthat the segmentsarose from replicative forks in which one of the branches was lost. The forks seenin Figure 1 were the only ones clearly displayed in this particular experiment. Although densely labeled segmentswere frequently seen, they occurred as linear non-forked configurations. This seemsto imply that the forks are fragile and tend to break at the point of replication when the DNA is being prepared for autoradiography. Fragility of the growing point has been observed in late intracellular phage h DNA by Skalka et al. (1972) and in E. co.&DNA by Hanawalt & Ray (1964). A problem encountered in interpreting structures observed in the autoradiographs of DNA labeled at two specific activities is to distinguish between individual densely labeled strands and clustered lightly labeled strands. This difficulty is largely avoided when only a single specific activity of thymidine is used to label just the newly replicated regions. (b) Lengths of pulse-labeledsegmentsof wild-type DNA A second experiment was performed in which only recently replicated DNA was labeled and the rest of the DNA was unlabeled. In this secondexperiment Casamino acids were omitted and the growth temperature was 37°C rather than 30°C. Wild-type phage-infected cells were labeled during the following periods: 10 to 15, 35 to 40 and 35 to 37.5 minutes. Although much of the labeled DNA observed in the autoradiographs was tangled or condensedas shown in Figure 2 (i), (j), (k) and (l), large numbers of relatively straight DNA segments were also clearly displayed. Length measurements were made of all well-displayed DNA segments obtained in the autoradiographs. DNA segments were considered well-displayed if their grain densities were relatively uniform along their length so as to minimize the likelihood of counting t’angled or condensed DNA. Examples of segments used in making measurements are shown in Figure 2 (a) to (f). In the wild-type infection a total of 1191 segments was measured from the three labeling periods. Most labeled segmentsmeasured were linear and therefore apparently only single branches of replicating forks. Only about I ‘I/, of the measured DNA structures were clearly V-shaped. The length distrilmtions of the segmentsfor each labeling period are shown in Figure 3 (a), (b) and (c). The median and mean lengths for each distribution are indicated in the legend to Pigurc 3. Since we had no way to distinguish unequivocally whether a given labeled segment was a single branch of a fork or both branches of a fork still connected, we put all measuredlengths obtained under a given set of conditions into a single histogram. It is informative to compare the length distributions of segmentsobserved in the &minute pulse from 35to 40 minutes (Fig. 3(b)) with thoseof the 2.5-minute pulse from

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FIG. 2. Linear tracks from wild-type-infected cells 40 min and (c) 35 to 37.5 min. DNA V structures for (d) 10 to 15 min, (e) 35 to 40 min and (f) 35 to mature phage particles; (i) and (j), condensed DNA condensed configurations of DNA. Scale bar represents

35 to 37.5

minutes

(Fig.

3(c)).

The

distribution

ET

AL.

pulse-labeled for (a) 10 to 15 min, (b) 35 to from wild-type-infected cells pulse-labeled 37.5 min. (g) and (h), DNA extracted from ring structures; (k) and (l), typical partially 50 pm.

of 2.5-minute

pulse-lengths

is plotted

pulse distribution. The shapes of the two distributions are very similar and the medians and means, as expected, differ by approximately twofold. The B-minute pulse-length distribution is more skewed towards higher values than the 2*5-minute pulse. This may reflect some running together of neighboring labeled segments occurring with the longer pulse. The mean length of the “10 to 15 minute”labeled segments was 29% greater than the mean length of the “35 to 40 minute” labeled segments. This may indicate a faster elongation rate or perhaps more frequent running together of labeled segments early in infection.

on a twofold

expanded

scale

for

ease

of comparison

to the

B-minute

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REPLICATION

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969

T4

(a)

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Length

tp m)

FIG. 3. Histograms of the lengths of autoradiographic tracks of pulse-labeled T4D+ and nmNG676 DNA. The phage strains used, the period during which [methyZ-3H]thymidine w&s added, the median and mean lengths of the observed tracks end the number of slides enalysed for each condition were, respectively: (a) T4D+. 10 to 16 min, 43.0 pm, 46.8 pm, 6 slides. (b) T4D+, 35 to 40 min, 29.6 pm, 36.3 pm, 6 slides. (c) T4D+. 36 to 37.5 min, 13.8 pm, 16.0 pm, 6 slides. (d) amNG676, 10 to 16 min, 41.3 pm, 43.2 pm, 5 slides. (e) (unNG676, 35 to 40 min, 43.6 pm, 47.2 pm, 4 slides. (f) amNG676, 70 to 76 min, 47.0 pm,, 61.7 pm, 6 slides.

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FIG. 4, Pairs of apparent replicative forks or loops. The phage strain from was obtained and the time of pulse-label are: (a) u~aNGS76, 36 to 40 min; (b) 76 min; (c) T4D, 10 to 15 min; (d) and (e) amNGS76, 70 to 75 min; (f) T4D, amNG678, 36 to 40 min. In the case of (a), (b) and (g) a diagrammatic interpretation labeled DNA is shown with the presumed associated unlabeled DNA indicated Scale bar represents 60 pm.

which the DNA anzNG676, 70 to 36 to 40 min; (g) of the pulseby broken lines.

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T4

9il

(c) Lengths of pulse-labeled segments of am mutant DNA The length distributions of amNG576 DNA are shown in Figure 3 (d), (e) and (f). A total of 719 segments was measured. As before, the measured tracks included a minority (about 3.5%) of V-shaped segments in the distribution. Our purpose in examining pulse-labeled lengths of DNA from this mutant was to determine if a DNA-delay am mutation would slow the rate of elongation. It is clear from Figure 3 t,hat the lengths of the segments labeled by 5-minute pulses in the DNA-delay mutant, are not shorter than the wild-type lengths labeled for 5 minutes. The length distributions of the “lOto 15”, “ 35 to 40” and the “70 to 75 minut)e” pulses of the am mutant, were very similar to each other as well as to the length distribution of the “10 to 15 minute” pulse of wild-type. These results imply that elongation is not slowed by the DNA-delay am mutation. Furthermore, it appears that the rate of elongation did not vary by more thanabout 20 o/oduring the course of infection by the DNA-delay mutant. (d) Replicutive forks and loops As pointed out above, in the autoradiographs of both wild-type and amNG576 DNA a number of clear V configurations were observed (see Fig. 2 (d), (e) and (f) and Fig. 4). About 1 y. of the wild-type and 3.5% of the am segments were V configurations. The mean length of the V structures from the B-minute pulse experiments with wildtype was 84.3 pm and from the experiments with the am mutant was 75.7 pm. The mean lengths of the linear segments were 42 pm for wild-type and 47 pm for the am mutant. Thus from their size and conformation most of the V configurations apparently represent undissociated replicating forks. About 20”,/0 of these fork structures were associated in pairs in which the two forks were within a few hundred pm of each other. All of these pairs are shown in Figure 4. The structures in Figure 4 (a) and (1)) appear to reflect bidirectional replication in a replicative loop that was already formed before addition of the label. Figure 4 (c) and (f) show two replicative forks apparently associated in a common structure and having the same direction of movement. The pairs of forks seen in Figure 4 (d) and (e) also appear to be part of a common structure because of their proximity to each other, but their relationship is unclear. The two loops shown in Figure 4 (g) apparently arose when two initiations occurred at nearby sites soon after addition of the label. (e) Calculution of rates of elongation in nucleotides per second When DNA is spread on a Millipore filter the number of nucleotide pairs per unit, length may be influenced by the filter surface. To determine the number of nucleotide pairs per unit length of phage T4 DNA on this surface the lengths of chromosomes extracted from mature phage were used as a standard. Lengths of phage chromosomes on the shiny sides of Millipore membranes were measured in autoradiographs of DNA released from mature phage by osmotic shock or treatment with perchlorate (Bernstein, 1970). The lengths of 387 chromosomes were determined and the distribution shown in Figure 5 was obtained. Examples of these chromosomes are shown in Figure 2 (g) and (h). The mean length was 36.8 pm and only 3% were longer than 45 pm. The methods employed for release of DNA from mature phage are harsher than those used for release of intracellular DNA, and fragmentation is expected. Jn selecting configurations on the autoradiographs for measurement, small broken fragments were avoided.

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60

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(pm)

FIG. 6. Histogram of the lengths of autoradiographic tracks of DNA extracted from mature phage. The median length was 36.9 pm and the mean length was 36.8 q. Five Millipore filters were scanned and 387 DNA tracks were measured. Short stretches of DNA were regarded as fragments and not included. Examples of measured tracks are shown in Fig. 2.

The molecular weights of phage T4 and T2 chromosomesare used to determine the number of nucleotide pairs per phage chromosome equivalent. The average of ten molecular weight determinations compiled by Bernstein (1970) and a more recent determination made by Klotz & Zimm (1972) was 1.20 x lo*. Using the average molecular weight per nucleotide of 342 for T4 and 336.5 for T2 (Bernstein, 1970), we can calculate that there are 1.77 x 105&O*09x lo5 nucleotide pairs per phage chromosome. In Table 1, column (6), the mean rates of replication in pm per minute, asdetermined from the histograms in Figure 3, can be expressedin chromosomeequivalents per minute (column (7)) by dividing each rate by the mean length of the mature chromosome (36.8 pm). These figures can then be converted to nucleotides per second by multiplying column (7) by 1.77 x lo5 and dividing by 60 to expressthe rate on a per second basis. We find that the rates of elongation of wild-type and amNG576 fall within the range of 516 to 829 nucleotides per second (Table 1, column (8)). As shown in Table 1, column (8)) the rate of elongation of wild-type T4 DNA at 37°Cappearsto be lower than the rate measured at 30°C in the first experiment. This may be due to the Casamino acids present in the media of the 30°C experiment. In addition, the rate of elongation at 30°C is based on the measurement of only two individual forks which fall within the 37°C distribution of Figure 3, so the apparent differences may not be significant.

E E L

E L L

(L) synthesis

(E) or linear

(3) Label given during exponential

256 133 330

379 388 424

Number of tracks measured

(4)

43.2 47.2 51.7

46.8 36.3 16.1

(rm)

Mean length of track

(5)

of

8.64 9.44 10.34

9.36 7.26 6.44

Rate

0.235 0.257 0.281

0.254 0.197 0.175

(r)t

of elongation (Chromosome equivalent min)

(7)

ph.uge T4 DNA

(6)

(&min)

pulse-labeled

1

693 768 829

749 581 616

$

S)

(Nucleotides/

(8)

0.034 0.034 0.035

0.14 0.14 0.14

@I§

Rate of DNA increase

(9)

0.14 0.13 0.12

0.56 0.71 0.80

k?)TI

Growing points chromosome equivalent

(10)

elongat,ion in chromosome equivalents/min (r) is obtained by dividing the rate in pm/min (column (6)) by the mean length of the phagc Millipore membranes (36.8 pm). elongation in nucleotides/s is obtained by multiplying the values in column 7 by 1.77 x lo5 (the number of base-pairs in a phage chromoby 60 (to convert to s). of k are derived from the DNA synthesis curves shown in Figures 6 (a) and (b) and 7(a) and (b) and as devcribcd in the t.ext.. The values equal to k, if they are exponential phase values and k, if they are linear phase values. phase values and gr, for linear phase values. of growing points/chromosome equivalent of template DNA is equal to gE for exponential which xvere obtained as described in thr trst. hero wore calculated from the relationships k, ~ r,.g, and k,~ = r,.g,,

l&15 36-40 70-75

amNG576

tThe rate of chromosome on $The rate of some) and dividing $The est,imates shown here are YThe number The values given

lo-15 36-40 36-37.5

Interval of pulse (min)

(2)

+

T4D

Phage strain

(1)

Characteristics

TABLE

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(f) The rates of DNA

increase during the exponential phases of wild-type infected cells

and linear

The course of DNA increase in wild-type phage-infected cells growing at 37°C under the same conditions as those in the pulse experiments, is shown in Figure 6(a) (semi-logarithmic plot) and (b) (linear plot). DNA increase starts at about 6 minutes after infection, proceeds exponentially until 17 minutes and then follows a linear

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(min) (b)

FIG. 6.(a) trichloroacetic This curve the amount normalized ulated and increase in exponential

DNA accumulation was measured as incorporation of [naethyL3H]thymidine into acid-precipitable material by the procedures described in Materials and Methods. represents the averaged results from 21 separate experiments. In each experiment of DNA accumulated at 36 min was set at 100 units and the counts at all other times against this value. At each of the times indicated the mean amount of DNA accumthe estimated standard error of the mean are given. This plot shows that the initial DNA is exponential with a doubling time (to) of 4.6 min, and that the transition from to linear increase occurs when about 29 units of DNA have been made.

(b) The data shown in (a) are replotted here on a linear scale. The curve shows that late DNA synthesis occurs at a linear rate (k,) of 4.0 units/mm The inset presents on an expanded scale the initial stages of synthesis including the transition from exponential to linear synthesis. Again we can see that the transition to a linear mode of synthesis occurs when about 29 units DNA have been made.

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course. It can be seen that an exponential curve (a straight line on a semi-log graph) followed by a linear curve (a straight line on a linear graph) joined at a point where t,heir slopes are just equal gives a total curve which accurately reflects the data. The doubling time during the exponential phase is 4.6 minutes. We can use the equation N, = N,exp(k,t) to represent the exponential increase of DNA during the eerly part of infection, where N, is the amount of DNA present at an arbitrary point and N, is the amount present after time t. The rate constant Ic, is expressed in “fold increase in DNA per minute”. To calculate the value of k, corresponding to a doubling time of 4.6 minutes the above equation is used with N,/N, sot> at 2. This gives a value for k, of 0*14-fold per minute. Exponential DNA replication can occur if the formation of new growing points keeps pace with the increase in DNA, so that the number of growing points per unit of DNA remains constant. Two possible models can be proposed to account for the subsequent shift to linear increase. On the one hand we can assume that exponential increase continues until all the replicative complexes, or all of some other cellular csonstituent required for formation of new growing points, become fully utilized. When this occurs, exponential increase gives way to linear increase with the number of growing points per cell remaining constant. On the other hand, it might be that the transition to linear synthesis occurs because of the onset of DNA packaging, which withdraws DNA from the replicative pool. To select between these alternatives we measured DNA increase with gene 33 and 55 am mutants which are unable to synthesize the late proteins needed for morphogenesis and DNA packaging (Hosada & Levinthal, 1968), but do synthesize DNA at normal rates. Our measurements showed t)hat here too DNA increase was first exponential and then linear. The transition from exponential to linear increase occurred at about the same time as in the wild-typeinfected cells (data not shown). This implies that the change to linear synthesis is not dependent on withdrawal of DNA from the replication pool by packaging. As described above, at about 17 minutes after infection by wild-type the increase in I)NA changes from exponential to linear. Linear increase can be represented by the csquation N = k,t, where N is the amount of DNA synthesized during time t, and k, is the rate constant in units of DNA per minute. The rate constant k, is equivalent to the slope of the linear portion of the curve in Figure 6(b) and is equal to 4.0 units I)NA per minute. The definition of a unit of DNA is given in the legend to Figure 6(a). On the basisof our experiments with gene 33 and gene 55 mutants and the shapeof the DNA increase curve, we postulate that the transition from exponential to linear increase occurs when an amount of DNA is synthesized which saturates the limited number of available replicative enzyme complexes so that the number of growing points ceasesto increase. The amount of DNA present at the time of transition is designated N, and is regarded as the amount of “template DNA” containing the final number of growing points. The value of Nf can be obtained from the transition point in Figure 6(a) and (b) which occurs at about 29 units of DNA. The rate constant k, (4.0 units/min) divided by N, (29 units) is the rate (kJ at which the template DNA forms new DNA. This has a value of 0.14-fold per minute. Since kE (the rate constant during exponential synthesis) and k, (the rate constant during linear synthesis) both equal 0.14-fold per minute it appears that the rate of DNA increase per chromosomeequivalent occupying the replicative complexes is constant throughout both the exponential and linear phasesof synthesis.

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(g) The number of growing points per chromosome equivalent of wild-type DNA

We would expect the exponential phaserate constant k, to be equal to the rate of elongation rs (in chromosomeequivalentslmin per growing point) times g,, the number of growing points per chromosomeequivalent. Similarly, in the linear phase k, should be equal to r,,the rate of elongation during the linear phase, times g,, the number of growing points per chromosomeequivalent present at the time of transition to linear synthesis. The values of rs and rL were obtained from the autoradiographs as described in section (e) above. The values of k, and k, were derived from Figure 6(a) and (b) as described in the preceding section. The calculated values of gs and gL are shown in Table 1, column (10). The number of growing points per chromosome equivalent for wild-type varies from 0.55 during exponential phase to 0.71 to O-80 during linear synthesis. (h) The number

of growing points per chromosome

equivalent

in amNG576 DNA

DNA increase in cells infected at 37°C by amNG576 is shown in Figure 7(a) (exponential plot) and (b) (linear plot). In the caseof the am mutant, just as in the wildtype infection, DNA accumulation can be accurately represented by an exponential curve (a straight line on a semi-log plot) followed by a linear curve (a straight line on

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(a)

00

100

20

120

Time

(min)

40

60

00

100

120

W

Fro 7(a). DNA accumulation by amNG676 (gene 52). The curve represents the results from 6 individual experiments. Each mutant experiment was run in parallel with a wild-type control. The amount of [methyl-oH]thymidine incorporated at 36 min by wild-type was set at 100 units and the mutant at each time-point was normalized against the wild-type 36-min value. At each of the times indicated the mean amount of DNA accumulated and the estimated standard error of the mean are given. This plot shows that at early times DNA increase is exponential with a doubling time of 20 min, and that the transition from exponential to linear increase occurs when about 66 units DNA have been made. (b) The data shown in (a) are replotted here on a linear scale. The curve shows that late DNA increase ocours at a linear rate (k,) of 2.3 units/min. The transition from exponential to linear increase occurs at about 66 units DNA.

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a linear plot) joined at a point where their slopes are just equal. The DNA doubling time during exponential synthesis was 20 minutes which corresponds to a k, value of 0.034-fold per minute. The rate of DNA increase during the linear phase (k,) is 2.3 units per minut)r and the amount of DNA at the transition point (N,) is 65 units. We can convert k, to k, if we divide lc, by N,. This gives a value for k, of 0*035-fold per minute. As with wildtype, k, and k, for amNG576 are about equal (0.034 and 0.035) but these rates are fourfold less than the corresponding rates of wild-type. From the values for rE and rL listed for the mutant in Table 1, column (7)) we can calculate g, (the number of growing points per chromosomeequivalent of DNA during exponential synthesis) and gL (the number of growing points per chromosome equivalent at the transition to linear synthesis). The calculated values, listed in column (10) of Table 1, are 0.14 and 0.13 for !yE and 0.12 for gL. We see that the number of growing points per chromosome equivalent is reduced in the mutant to 15 to 25% of the wild-type values.

4. Discussion (a) Accuracy

of estimated

elongation

rates

We have reported here estimates of elongation rates of phage T4 DNA based on direct measurements in autoradiographs of pulse-labeled DNA segments. These measurements are subject to several possible sources of error. Underestimates of elongation rates could occur by breakage or by recombination of the pulse-labeled segments, or by slow equilibration of nucleotide pools after addition of labeled thymidine. On the other hand, overestimates of elongation rates could occur by joining of separately initiated tandem replicating sequences. Breakage or recombination would be more likely to shorten the 5-minute pulselabeled segmentsthan the 2.5-minute segments,although if these processeswere very ext.ensive they would tend to fragment both types of segment to a common small average size. 9 delay in incorporating labeled nucleotides into DNA would shorten the 2.5-minute labeled segmentsrelatively more than the 5-minute labeled segments. Joining of tandem sequencesshould lengthen the 5-minute pulse-labeled segments more than the 2.5-minute labeled segments,since the longer segmentswould tend to run into each other more frequently. The mean length of the segmentspulse-labeledfrom 35 to 37.5 minutes was 16.1 pm, and from 35 to 40 minutes was 36.3 pm. The B-minute labeled segmentsare thus about twice the length of the 2.5-minute labeled segments.Furthermore, as shown in Figure 3(b) and (c), the distributions of lengths in the two caseswere similar after adjusting for the twofold difference in labeling period. These results therefore suggest that our estimates of elongation rates are largely free of the sourcesof error discussed although the finding that the B-minute pulse-lengths are 12% longer than twice the 2.5-minute pulse-lengths indicates somerunning together of pulse-labeledsegments. In the autoradiographs from the first experiment in which all of the DKA synthesized after infection was labeled, the spread-out DNA was typically in the form of long concatemers. This suggeststhat fragmentation of the pulse-labeled segments should not have been a major problem in the subsequent pulse experiments where the procedures for handling extracted DNA were as gentle as in the first experiment. We observed in Results (section (b)) that few of the pulse-labeledsegmentsappeared to be intact forks with both branches associated. However. to the small extent that

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intact forks were included among the measured segments, average elongation rates would have been overestimated. As shown in Figures 1 and 4 and noted in Results, about 20% of observed pulse-labeled forks were located close to a second fork of similar length. We infer from the similarity in dimensions of the two forks in each pair that neither was disrupted by breakage or recombination. These forks, as expeuted, when measured through both arms had about twice the mean length of the overall population of segments. In this collection of paired forks of wild-type and the am mutant the mean lengths were 94 pm and 87 pm, respectively, compared to the mean lengths of the corresponding segments of 42 pm and 47 pm, respectively. This suggests that estimates of elongation rates based on total numbers of segments were not greatly biased by breakage, recombination or the inclusion of a small proportion of intact forks. In the 10 to E-minute pulse experiments with wild-type and amNG576 the elongation rate may have been underestimated for the following two reasons. New initiations arising during a pulse-labeling period add short labeled segments to the population of segments from which the elongation rate is calculated. This bias would be greatest in early pulse periods because relatively few established growing points are present at the start of labeling. Also, if the DNA during the 10 to 15 minute labeling period was still in the form of single chromosomes and had not yet entered concatemers, elongation would tend to terminate at the end of the chromosome rather than continuing until the end of the labeling period. With regard to the latter problem, our evidence suggests that much (if not all) of the DNA at 10 to 15 minutes is in the form of concatemers. It can be calculated from the data in Figure 3(a) and (d) and Figure 5 that 47% of wild-type B-minute labeled segments and 44% of amNG576 segments are longer than 45 pm, but that only 3% of mature phage chromosomes are longer than this. Also, if either of the above biases were of substantial magnitude, we might expect that elongation rates determined during the early periods would be much less than those during later periods. However this is not so, as can be seen in Table 1, column (8). (b) Comparison of our estimates of DNA rates with other estimates

elorzgation

The rate of phage T4 DNA elongation has been estimated in vivo (Werner, 1968a,b) and in vitro (Morris et al., 1975). Werner’s estimate of elongation rate was 0.08 chromosome equivalents per minute, which is less than half the rates determined by us for wild-type (0.175 to O-254 as shown in Table 1, column (7)). However, since our infections were carried out at 37°C and Werner’s were done at 25”C, the different rates may not be inconsistent. Morris et al. (1975), using a cell-free system containing the purified products of six phage T4 genes and a temperature of 3O”C, obtained an elongation rate in vitro which was reported to be about 800 nucleotides per second. This estimate is in reasonable accord with our i?b vivo estimates for wild-type, which vary from 516 to 749 nucleotides per second. In E. wli, DNA elongation rates were measured in autoradiographs by Kuempel et al. (1973) and by Rodriguez & Davern (1976). The estimate of Kuempel et al. (1973) was 16.5 pm per minute. Rodriguez t Davern (1976) estimated a maximal rate of elongation in glucose minimal medium at 25°C of 13.6 pm per minute based on the longest pulse-labeled segments observed. However, the elongation rate based on all 98 pulse-

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labeled segments was 6.5 pm per minute. The E. coli elongation ra,te can a,lso he calculated indirectly from the chromosome transit time. which is the time required to replicate from the chromosome origin to the terminus. Values of 40 minutes (Bird 81 Lark, 1968) and 65 minutes (Pritchard 6 Zaritzky, 1970) have been determined in P178 a low thymine requiring strain of E. coli 15T-. The contour lengths in autoradiographs of 14 E. coli chromosomes were measured by Rodriguez et al. (1973). The mean length was 868 pm, although individual values varied over a wide range. Using this mean length and the two transit times given above. the elongation rate can be calculated to be bet,ween 7 and 11 pm per minute per fork if one also assumes that replication is bidirectional (Rodriguez et al., 1973 ; Prescott t Kuempel, 1972). The elongahion rates we det’ermined for wild-type phage T4 varied from 6.4 to 9.4 pm per minute (Table 1. column (6)). Thus our estimated rates are in reasonable accord with most of the E. coli castima,tes. These E. coli and phage T4 DNA elongation rates can be directly compared, since the number of nucleotides per unit length on Millipore membranes is vcr! similar for the two organisms (Bernstein, 1970). In eucaryotic cell lines the rate of DNA elongation appears to be slower t(han in E. colt’ or phage T4. For instance, in a recent autoradiographic st’udy Housman & Huberman (1975) found the rate of elongation in Chinese hamster ovary cells to rar) during S phase from 0.2 to 0.6 pm per minute. (c) G-owing

poind

distribution

In our a,utoradiographs we found no evidence that growing points are separated by distances of less than O-2 of a chromosome equivalent length as proposed by Werner (1968a,b). Where we have observed two neighboring forks (Figs 1 and 4) the growing points were usually a chromosome equivalent length or more apart (Fig. 4(d) is t’ht, exception). The finding that the forks in Figure 1 have grain densities expected from heavy labeling in just one DNA strand, as would result from simple semiconservativr replication, also argues against clustering of growing points. If growing points followed each other at close intervals, one or b0t.h of the individual arms in each fork should have been heavily labeled in both strands of their DNA. Although the evidence obtained by Werner (1968a,b) indicated that growing points u’ere closely clustered, he found the average number of growing points per chromosome equivalent of DNA to be only 1-O to 1.5. These values together with our estimat#es of 0.55 and 0.80 indicate that the number of growing points per chromosome equivalant of template DNA is close to unity. From the autoradiographs we can not tell if DNA replication is usually unidirectional or bidirectional. Configurations expected for unidirectional synthesis (forks traveling in the same direction, Fig. 4(c) and (f)) and bidirectional synthesis (forks traveling in opposite directions, Fig. 4(a) and (b)) were both found. The initial round of phage T4 DNA replication has been extensively studied (for a review see Miller, 1975). Evidence has been obtained that replication is initiated in a bidirectional manner at several specific sites per chromosome (e.g. Howe et al., 1973). However. other results (e.g. Marsh et al., 1971) show that there is a preferred site for initiation close to gene 43 and that replication from this site is unidirectional. OUI evidence and that of Werner (1968a,b) indicates that however many growing points per chromosome equivalent are present at the start of synthesis, the number a,pproaches unity in template DNA during subsequent rounds of replication.

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(d) DNA

MCCARTHY

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synthesis by a DNA-delay

mutant

We have concluded that in the infections by the DNA-delay mutant amNG576 the slow rate of DNA increase is due to a deficiency in growing points per unit of DNA rather than to a slow rate of DNA elongation at each growing point. By determining rates of DNA increase from the kinetic data (Figs 6 and 7) and rates of elongation from the autoradiographic data (Fig. 3) we have calculated that the number of DNA growing points is from 0.55 to 0.80 per chromosome equivalent of template DNA in wild-type but only 0.12 to 0.14 in the am mutant. To account for the apparent deficiency of growing points in the mutant DNA we postulate that the mutant is defective in the initial formation of growing points, presumably in the interaction of a DNA initiation sequence with a replicative enzyme complex. The result reported by Morris et al. (1975) that normal rates of DNA elongation can be obtained in an in vitro system containing the products of six phage T4 genes, but not including the products of the four DNA-delay genes, implies that the slow rate of DNA increase observed in the DNA-delay mutants is due to a defect other than in elongation. Naot Jr Shalitin (1973) have shown that a DNA-delay am mutant defective in gene 52 allowed expression of a nucleolytic activity not present in wild-type and proposed that control of this activity is essential for proper initiation of DNA replication. (e) The functions

of the DNA-delay

genes

In addition to the evidence considered in the preceding section suggesting a defect in the formation of growing points, other lines of evidence also add to our understanding of the DNA-delay gene functions. Huang (1975) has shown that the proteins specified by DNA-delay genes 39 and 52 are specifically associated with the host cell membrane. DNA-delay mutations have also been shown to cause alterations in the membrane transport of Mg2+ (G u tt man BE Begley, 1968) and spermidine (Dion & Cohen, 1971). Intracellular phage DNA is prematurely released from its membrane association in gene 52 am mutant-infected cells followed by subsequent reattachment The ability of all DNA-delay am mutants to grow on su(Naot & Shalitin, 1973). E. wli hosts at 37°C was investigated by Mufti & Bernstein (1974). Evidence was obtained that this growth was due to a host cell component which can partially compensate for the lack of the DNA-delay gene products, and that the wild-type products of the DNA-delay genes interact with the host component to adapt the cell to phage DNA synthesis. Huang Q Buchanan (1974) found that the products of the DNA-delay genes 39 and 52 bind specifically to DNA. DNA-delay am alleles have been shown to stimulate frameshift and base substitution mutation (Mufti & Bernstein, 1974) as well as recombination (Berger et al., 1969; Mufti & Bernstein, 1974; Leung et al., 1975). The individual strands of the replicative DNA produced by these am mutants have been shown to be shorter than the individual strands of wild-type DNA (Naot & Shalitin, 1972). Broker (1973) showed that DNA-delay gene 58-61 am mutants enhanced the occurrence of extensive single-stranded regions in the DNA of phage having additional ligase and DNA polymerase defects. These several observations indicate that in DNA-delay am mutant infections there is an increased tendency to produce altered or damaged DNA. Although there is no assurance that all DNA-delay mutant phenotypes are expressed through similar mechanisms, a tentative picture can be formulated. The

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DNA-delay gene products may enter a complex at the cell membrane which includes a host component. These gene products adapt the complex to allow efficient’ initiat,ion of growing points in phage DNA and, although they are not, necessary to maintain a norma,l rate of elongation, they are necessary for accurate replication. We thank Thomas North and script. This work was supported and by the National Foundation

William Howe by the National (grant l-399).

for their Science

helpful criticism of the manuFoundation (grant, GB8760A)

REFERENCES Adams, M. H. (1959). Bacteriophages, Interscience, London. Berger, H., Warren, A. J. & Fry, K. E. (1969). J. v’irol. 3, 171- 175. Bernstein, C. (1970). Biophys. J. 10, 1154-1172. Bernstein, C. & Bernstein, H. (1974). J. Viral. 13, 1346-1355. Bernstein, H. & Bernstein, C. (1973). J. Mol. Biol. 77, 355-361. Bird, R. & Lark, K. G. (1968). Cold Spring Harbor Symp. Qua&. BioZ. 33, 799-808. Broker, T. R. (1973). J. Mol. BioZ. 81, 1-16. Dion, A. S. & Cohen, S. S. (1971). J. Viral. 8, 925-927. Epstein, R. H., Bolle, A., Steinberg, C. M., Kellenberger, E., Boy de la Tour, E. & Chevalley, R. (1963). Cold Spring Harbor Symp. Quant. Biol. 28, 375-394. Guttman, B. S. & Begley, L. (1968). Virology, 36, 687-690. Hanawalt, P. C. & Ray, D. S. (1964). Proc. Nut. Ad. Sci., U.S.A. 52, 125-132. Hosada, J. & Levinthal, C. (1968). Virology, 34, 709-727. Housman, D. & Huberman, J. A. (1975). J. Il/loZ. BioZ. 94, 173-181. Howe, C. C., Buckley, P. J., Carlson, K. M. & Kozinski, A. W. (1973). ./. 1UroZ. 12, 130--148. Huang, W. M. (1975). Birology, 66, 508-521. Huang, W. M. $ Buchanan, J. M. (1974). Proc. Nat. Acad. Sk., U.S.A. 71, 2226.-2230. Klotz, L. C. & Zimm, B. H. (1972). J. Mol. BioZ. 72, 779-800. Kuempel, P. L., Prescott, D. M. & Maglothin, P. (1973). In DNA Synthesis in Vitro (Wells, R. D. & Inman, R. B., eds), pp. 463-472, University Park Press, Baltimore. Leung, D., Behme, M. T. & Ebisuzaki, K. (1975). J. Viral. 16, 203-205. Marsh, R. C., Breschkin, A. M. & Mosig, G. (1971). J. ,Wol. BioZ. 60, 213-233. Miller, R. C., Jr (1975). Anna Rev. Microbial. 29, 355--376. Morris, C. F., Sinha, N. K. & Alberts, B. (1975). Proc. :V’at. .1cad. Sci., l’.S.B. 72, 4800-. 4804. Mufti, S. di Bernstein, H. (1974). J. Viral. 14, 860-871. Naot, Y. & Shalitin, C. (1972). J. Viral. 10, 858-862. Naot, Y. & Shalitin, C. (1973). J. Viral. 11, 862-871. Prescott, D. M. & Kuempel, P. L. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 2842--2845. Pritchard, R. H. & Zaritsky, A. (1970). Nature (London), 226, 126131. Rodriguez, R. L. & Davern, C. I. (1976). J. Bacterial. 125, 346-352. Rodriguez, R. L., Dalbey, M. S. & Davern, C. I. (1973). J. Mol. BioZ. 74, 599-604. Skalka, A., Poonian, M. & Bart,l, P. (1972). J. 1MoZ. BioZ. 64, 541-550. Steinberg, C!. M. & Edgar, R. S. (1962). Genetics, 47, 187-208. Werner, R. (1968a). J. Mol. BioZ. 33, 679-692. Werner, R. (1968b). Cold Spring Harbor Symp. Quad. BioZ. 33, 501-507. Yegian, C. D., Mueller, M., Selzer, G., Russo, V. & Stahl, F. W. (1971). Virology, 46, 900-919.