Replication of bacteriophage λ DNA dependent on the function of host and viral genes

J. Mol.

Biol.

(1973) 75, 155-212

Replication of Bacteriophage h DNA Dependent on the Function of Host and Viral Genes I. Interaction L.

W.

of red, gam and ret

ENQUIST

AND A. SKALEA

Roche Isstitute of Molecular Biology Department of Cell Biology Nutley, N.J. 07110, U.S.A. (Received 4 Xeptember 1972, and in revised form 29 December 1972) We have stud&d the role of the red and gam genes in lambda replication, after infection of wild type and two recombination deficient hosts. Our results show that the rate of phage DNA replication is abnormally low in the absence of red function, in ret+ as well as ret- (A- and A-B-) bacteria. It appears that the virus general recombination proteins play some role in lambda replication that cannot be assumed by the general recombination proteins of its bacterial host. The red- defect in replication results in a decrease in the total amount of intracellular phage DNA. This DNA, nevertheless, seems normal in structure and is matured and packaged with good cffioioncy. hosts infected with gam- mutants, the rate of lambda In ret+ and recAreplication is also low, but in this case, abnormal DNA structures are produced at late times. The gum mutation seems to alter the program of replication such that circular molecules are produced not only at early times, but continuously, throughout the lytic cycle. This, and other facts, suggest that the gana protein is This requirement required for the transition from “early ” to “late ” replication. for gun% function is not observed in recA - B- hosts, in which gum mutants replicate at a normal rate and produce DNA indistinguishable from that made by wild type phage. Thus, the gam requirement seems to involve an interaction of this pha,ge protein with the product of the host’s recR gene. Other evidence for such interaction comes from our finding that, in &JO, the gum protein does inhibit presumed action of the host’s BC nuclease. In t’he garn- mutant infections, which are blocked in late replication, absence of a general recombination system seems to create a severe defect in maturation of intracellular phage DNA. This defect, unlike the one affecting A replication rate, oan be alleviated by either the red or ret functions and is correlated with the inability of the mutant phagcs to make DNA concatemers. Since other late functions (i.e. late messenger RNA production) appear to be normal, we conclude that concatemer formation, via replication or recombination, is an essential step in phage development.

1. Introduction A new h gene, gamma (gam), was recently discovered by Zissler et al. (1971). These authors surmised that the gum gene product was “associated in some way with recombination ” because gum- mutants were slightly defective in U.V. repair and recombination and because they failed to grow in hosts lacking DNA polymerase (polA-). Most pertinent to this report, however, was the observation that the gum 13

185

186

L.

W. ENQUIST

AND

A. SKALKA

gene product was required for red- phages to plate on a recombination-deficient recA- host (“fee- ” for “feckless” because of the failure to plate on recA- hosts; Manly et al. 1969). Zissler and co-workers showed that this requirement was relieved by a defect in the host’s recB gene, since red-gam- phages did plate on recA-Bhosts. Our study of gam gene function stems from an interest in the origin and role of A DNA concatemers that are produced at late times during normal replication. In order to distinguish between two possible origins of concatemers, i.e. via recombinaphages growing in tion or replication, a system had been constructed (red-idrecA- bacteria) that was defective in the production of genetically recombinant phages (Skalka, 1971a). In this system, DNA concatemer formation occurred normally (Skalka et al., 1972). It was concluded that concatemers originated mainly via replication (this was considered most likely) or by some hypothetical recombination system of no genetic consequence. The discovery that a mutation in the gam gene could make the red-int-retsystem inviable led us to suppose that the gam function might play an important role in replication. The following report summarizes our analyses of DNA synthesis by gam- and red- mutants. A preliminary report of these findings has been published (Enquist & Skalka, 1972).

2. Materials and Methods (a) Bacterial a& phage strains Escherichia coli 204 thy- recA- SU- was obtained from M. Me&son. E. coli KL254 recA1 recB21 su+ was obtained from M. Oishi. E. coli W3110 thy- szc-, a strain related to 204, was used as the ret+ control. Plaque counts were made on E. co& C600 (edI+), W3350 (8~~) or QD5003 (suLII+), obtained from Ethan Signer. The phages used were h betam cI857, Ji betam gamam210 ~1857, X gamam210 ~1857, X pbioll ~1857, h pbio72 ~1857, h betl13, X gum5 and h bet113 gam5, all from J. Zissler. Lambda ~I857 Sam7 was obtained from Ethan Signer. The betam mutation is polar and reduced activity of the exe gene product (J. Zissler, personal communication) and bet1 13 is a missense

TABLE 1 Recombination frequencies in the h map interval OP to S for various recombination-defective mutants Mutation

Bacteria 9W+

recA -

recA- B-

A red-

113 (bet-) am270(bet-)

0.83 1.7

0.05 0.02

0.03 -

h gana-

5 am210

1.0 0.83

1.9 0.2

1.5 -

X red-gam-

113,5 am270, am210

0.57 0.75

0.01 0.02

0.02 -

3.0, 2.9

2.3, 1.2

A Wild type

2.4, -

Bacteria (at a cell density of 1 x lO*/ml) were infected at a multiplicity of infection of 6 for each genotype. Each cross included X bio 10 (V h att-gam) Oam29 Pam80. The other input genotype aontained mutations listed above plus Sam7. Progeny phage were scored by plating on C600 (surI*) and QD5003 (szcrII+). Recombinants were scored by plating on W3350. Numbers in the Table are plaques on W336O/(plaques on C600 + QD5003) x 100.

187

h REPLICATION

mutation in beta (Signer et al., 1968). Unless otherwise specified, red and gum amber point mutants were used. When desired, the S mutation or an dam11 (from a mutant in our collection) was crossed into phage stocks by conventional techniques. The recombination defects in the recA- and reck - B- bacteria and all of the phage stocks were verified by appropriate crosses, in which the frequency of recombination in the interval OP to S was measured. Results obtained (Table 1) were consistent with published data. The presence of the recA mutation in the recA - B- strain was verified by tests of U.V. sensitivity and the presence of the recB mutation by checking for the “cautious” phenotype (Willets 85 Clark, 1969). Hereafter, all mutant number designations will be omitted in the text. Since most phages contained the ~I857 mutation, and its defect is not pertinent to these studies, that designation will also be dropped from the text and phage carrying only the ~I857 mutation will be called wild type. The locations of the various A genes and deletions relevant to this work are shown in Figure 1. Left

Right arm

arm

A

b,

J

Heads

Talk

“Silent”‘* r-F-

c7” I

Cl Recombination

CC*-

I I

OP 5-R II II II II ‘\DNA synth. Lysis ‘\ \

**--

--

- y

bio 72 subslitution

’

1

j

, I

I

I I

t I

,

---

FIG. 1. Genetic and functional map of bacteriophage lambda and relevant mutant phages. The recombination region has been expanded (not to scale). The approximate extent of the bio substitution mutations are indicated by shaded regions below the expanded portion. (b) Media Minimal medium (Joyner et al., 1966) supplemented with 0.2% vitamin-free Casamino acids was used in all experiments. Plating lysates were grown in Tryptone broth. (c) Prelabeling

of host DNA

maltose bacteria

and 0.02% and phage

with [14C]thymidine

After dilution of an overnight culture with fresh medium to a concentration of about lo7 cells/ml, d[14C]Thd ([methyl-14C]thymidine, 55.3 mCi/mmol, from New England Nuclear) was added to give a final concentration of 1 to 2 $X/ml. When the cell density reached 2 x lOe/ml, the cells were harvested by centrifugation and washed twice with 2 vol. ice-cold medium without thymidine. The washed cells were then suspended at a density of 4 x log cells/ml in O-02 M-MgSO, and used for standard infection experiments (Skalka, 1971a). Unless specified otherwise, all infections were at a multiplicity of infection of 0.5 to 1.5. (d) Preparation of 3H-labeled DNA Labeling of DNA in recA - -infected cells has been described previously (Skalka, 197 la), Similar methods were used to label recA-B--infected cells. The recA- B- bacteria, like recA- cells, are extremely sensitive to low doses of U.V. light (Willets & Clark, 1969). By treatment with 350 erg u.v./mm 2, host DNA synthesis was reduced to negligible levels and colony-forming ability was reduced better than 99.99%. Because the recA-Bcells were not thymine requirers, thymine was omitted from the labeling medium. Sufficient uptake of [3H]thymidine was obtained by maintaining its concentration at O-5 pg/ml.

188

L. W.

ENQUIST

AND

A. SKALKA

(e) Measurement of trichloroacetic acid-precipituble counts Samples (0.250 ml) were placed on ice and made 5% in trichloroacetic acid by the addition of an equal volume of ice-cold 10% trichloroacetic acid containing 2 mg unlabeled dThd/ml. Two drops of 0.25% bovine serum albumin were added and the resulting precipitate pelleted by centrifugation at 3000 revs/min in the Sorval RC2 centrifuge. The supernatant was removed and the pellet dissolved in 0.5 ml of 1 y. NH&OH; 4 ml ice-cold 5% triehloroacetic acid was then added and the resulting precipitate was pelleted again. After removing the supernatant, 0.5 ml of 1 N-NaOH was added and the solution incubated at 37°C overnight. After incubation, the solution was neutralized with 1 NHCl; 2 drops of 0.25% bovine serum albumin and 4 ml ice-cold 10% trichloroacetic acid with 2 mg unlabeled dThd/ml were added and the resulting precipitate collected by in 0.5 ml of 1% NH,OH and precipitated with centrifugation. The pellet was dissolved 4 ml of 5% ice-cold trichloroacetic acid. After centrifugation, the pellet was dissolved in O-5 ml NCS solubilizer (Amersham-Searle Corp.), heated at 60°C for 5 min and counted in Spectrofluor (Amersham-Searle Corp.) scintillation fluid containing 2 ml glacial acetic acid/l. (f) Extraction of labeled phage DNA The technique of Skalka ( 197 la) was employed, except for the following modifications : lysozyme at final concn of 0.8 mg/ml was used to aid lysis. Immediately after the Sarkosyl and pronase additions, the DNA solution was heated to 65°C for 5 min and then cooled on ice. This solution was incubated at 37°C for 1 to 2 h, heated to 65°C for 5 min a second time and then dialyzed overnight in the cold Ver8ZL.sthree l-l changes of 0.05 M-Tris buffer (pH 8.0) and 0.01 X-EDTA. (g) ll!ireasurement

of irzitial

rates of d[“H]Thd

incorporation

Samples (0.250 ml) of infected cells were taken into an equal volume of warmed medium with enough d[3H]Thd (meth$-3H, 10 to 15 Ci/mmol; New England Nuclear) to give a final concentration of 10 &X/ml. For recAinfections, thymine was added to 5 pg/ml. For recA- B- infections, dThd was added to 0.5 pg/ml. For ret+ infections, the final specific activity of d[3H]Thd was 0.3 pCi/pg dThd. After 1 min (at 46 or 37”C, as indicells, incorporation was cated) for the recAand ret+ cells or 2 min for the recA -Bstopped by the addition of 1 ml ice-cold 10% trichloroacetic acid containing 2 mg unwas then treated as described in section (e) labeled dThd/ml. The resulting precipitate above. (h) Equilibrium centrifugation in CsCl containing ethidium bromide Supercoiled covalently closed DNA molecules were separated from open circular and linear DNA molecules by equilibrium centrifugation in CsCl solutions containing ethidium bromide (Bauer & Vinograd, 1968). Saturated CsCl (3.0 ml) was mixed in the centrifuge of DNA, and ethidium tube with 0.050 ml of 1 M-Tris buffer (pH 7.4), a l-4-ml solution bromide (500 pg) (a gift from Boots Pure Drug Co., England). The refractive index was adjusted to l-3887 and the solution covered with mineral oil. The Spinco 50Ti rotor was used and centrifugation was at 43,000 revs/min for 27 h at 30°C. Fractions were collected from the bottom of the tube on paper discs, which were then dried and washed 3 times in ice-cold 5% trichloroacetic acid. The discs were finally washed in 95% ethanol, dried and assayed for radioactivity. (i) Enzymic

detection

of nicks and gaps in circular

DNA

molecules

(i) Enzymes and substrates Nuclease-free phage T4 polynucleotide ligase, T4 DNA polymerase and T4 polynucleotide kinase were generous gifts of Dr C. Harvey, Hoffman-La Roche Inc. The polynucleotide ligase preparation had a spec. act. of 6000 units/mg (1 unit = 1 nmol 32P exchanged in 20 min; Weiss et al., 1968). The activity of the DNA polymerase preparation was 30,000 units/mg (1 unit = 10 nmol total nucleotides made acid-insoluble in 30 min at 37’C using kinase activity was denatured single-stranded DNA; Go&an et al., 1968). Polynucleotide in 30 mm; Richardson, 1965). Hydro20,000 units/mg (1 unit = 1 nmol 3aP incorporated gen-bonded circles were prepared from linear h DNA as described by Gellert (1967).

189

h REPLICATION

(ii) Reaction mixture The reaction mixture (0.125 ml) contained 40 mM-Tris (pH 7*4), 1.4 mM ATP, 0.2 mM each of dGTP, dATP, dTTP and dCTP, 5 mM-MgCl,, 0.03% bovine serum albumin, 1 nmol of nucleotides in 32P-labeled, hydrogen-bonded circular X DNA and less than 0.10 nmol of nucleotide in 3H-labeled DNA. The 3H-labeled DNA (in 0.01 ZM-Na+) was heated for 3 mm at 64*5”C and then cooled immediately before addition to the reaction to eliminate any end to end aggregation. For repair of nicks, 0.5 unit of polynucleotide ligase was used. For repair of gaps, 0.5 unit of DNA polymerase was included with 0.5 unit of polynucleotide ligase. All reactions included O-5 unit polynucleotide kinase. The reaction mixture was incubated at 25% for 15 min, and then stopped by addition of EDTA (final concentration, 96 mm). (iii) Assay of covalent closure After addition of EDTA, the entire reaction mixture (0.18 ml) was layered on a 5-m 5 to 20% sucrose gradient, containing 0.7 M-NaCl, 0.3 M-NaOH and 1 mu-EDTA, formed over a O-4-ml bottom pad consisting of 70% sucrose and 80% sodium iothalmate (AugioConray, Mallinckrodt Pharmaceuticals) mixed 1: 1 and adjusted to 0.3 M-NaOH. Gradients were centrifuged for 120 min at 45,000 revs/mm in the SW 50.1 rotor at 11°C. Under these conditions, linear molecules sedimented halfway down the gradient, and covalently closed circles, which sediment at about 3.5 times the rate of linear single-stranded molecules, accumulated in the dense bottom pad. To measure accurately the sedimentation rate of oovalently closed duplex linear molecules, the same conditions were used, but the centrifugation time was decreased to 100 min. Fractions were collected directly into vials oontaining buffered scintillation fluid.

3. Results (a) Synthesis of DNA

in. recA- bacteria infected with red-, and wild-type phage

gam-,

red-gam-

Figure 2 shows the results of measurements of the incorporation of d[3H]Thd into trichloroacetic acid-insoluble material in u.v.-irradiated, infected and uninfected

r----’

0

X red-gum‘

d

d-d-*-

20

40

60

80

Time after infection hn)

FIG. 2. Incorporation of d[3H]Thd into trichloroacetic acid-insoluble material during infection of recA - bateria. ( A) Infected with wild type; (0) infected with red- ; (0) infected with garn- ; (a) infected with red-gam- ; ( x ) uninfected cells. Infected u.v.-treated cells were suspended in 5 ml minimal medium (plus 5 pg thymine/ml) containing 40 @i d[3H]Thd/ml, incubated at 46°C for 5 min with aeration, followed by incubation and aeration at 37°C for the duration of the experiment. Samples (0.250 ml) were taken at indicated intervals and the trichloroacetic acid-insoluble radioactivity determined as described in Materials and Methods.

190

L.

W. ENQUIST

AND

A. SKALKA

recA- bacteria. During the normal growth period (0 to 40 min), both the red- and gam- mutants accumulated only about half the amount of DNA made by the wild type phage. The defect, most apparent late in infection, seemed even greater with the red-gam- double mutant. However, here and in all the following experiments, the amount of red-gam- DNA is somewhat underestimated because added isotope is diluted by the pool of nucleotides, which results from the breakdown of bacterial DNA (see section (d) below). Analogous results were obtained with deletion mutants X pbio72 (red-) and A pbioll (red-game) and the data are not shown. As has been shown previously (Skalka, 1971a), uninfected cells incorporate negligible isotope following u.v. irradiation as used here. (b) Comparisons of production of phage and phage equivalents of DNA To verify that the trichloroacetic acid-insoluble radioactivity measured as described in Figure 2 was lambda specific, samples were annealed with nitrocellulose membranes containing an excess of X DNA. DNA content, expressed as phage equivalents per infected cell, was calculated from the amount of acid-insoluble radioactivity tha,t hybridized to X DNA, the specific activity of [3H]thymine in the medium and the known thymine content of h DNA (Carter & Smith, 1970). The results (Fig. 3) demonstrate the effect of red- and red-gam- mutations on the produotion of h-specific DNA and free infectious phage particles. These data are qualitatively similar to those in Figure 2. In all instances, there was a 30 to 60-fold

I

X Wild type

/

X Wild type /

l -t

IO

20

30

Tome after

40

50

60

infection

FIG. 3. Synthesis of free phage and phage equivalents in intracellular DNA during infection of Open symbols indicate phage equivalents of DNA per infective center. Closed symbols are plaque-forming units per infective center. Circles, infected with wild type; squares, infected with red- ; triangles, infected with red-gam-. The infected cells were suspended in 6 ml minimal medium containing 10 pCi [3H]thymine/ml (methyl labeled, 11.7 Ci/mmol; New England Nuclear). Unlabeled thymine was added to give a spec. act. of 2.2 @i/pg. Samples (0.2 ml) were taken at the indicated intervals and precipitated with trichloroacetic acid as described in Materials and Methods. The amount of lambda-specific DNA in the precipitate was determined by DNA hybridization as described by Skalka (1971~) and Skalka 87 Hanson (1972). The DNA content of the phages was taken as 5.08 x IO-l1 pg and the content of thymine per lambda DNA molecule was estimated at 4.8 x IO-la pg. (The calculation of DNA phage equivalents was then determined as discussed by Carter & Smith (1970).) Plaque-forming units were determined by standard plaque assay using E. coli C600 (szlll+) as the indicator bacteria. The number of infectious centers was determined at 6 min after infection by plating a suitable dilution on E. coli C600.

reoA- bacteria.

191

X REPLICATION

net synthesis of DNA over and above that of the input phage (multiplicity of infection = 1). The ratio of free phage to intracellular DNA phage equivalents at 50 minutes after infection was 0.8 for wild type (average burst size, 67), O-6 for red(and yam- in results not shown) and about 0.1 for red-yam- phage. Although red-, yam- and red-yam- phage are apparently all defective in DNA synthesis, only the double mutant seems unable to express what DNA there is as plaque-forming units. Virtually identical results were obtained in control experiments with infected recAbacteria with no U.V. treatment. (c) Initial

rates of thymidine incorporation in infected ret + , recA- ati recA - B - hosts Measurements of DNA synthesis in the ret’ and recA- hosts after infection with the double mutant, red yam, or phage containing single mutations in either gene (Fig. 4(a) and (b)) show rates that are significantly lower than with wild type infection. When experiments to measure rates were conducted with a u.v.-treated recA-Bhost, results different from those found in ret+ and recA- cells were found. Here the yam mutation caused no change in the rate of DNA synthesis (Fig. 4(c)). The red mutation, however, did cause a significant decrease in the rate of synthesis. The red - gam- results are indistinguishable from those with red- alone. Appropriate control experiments indicated that these results, too, were independent of the U.V. treatment. In an attempt to determine if the observed rate defects were specific for “early” or “late” replication, the following experiment was performed. Thymidine incorporation was measured during one-minute “pulses ” at 2-minute intervals from zero

0

0

15

30

45

0

15

30

45

0

15

30

45

Time after infection (min)

Fm. 4. Initial rates of incorporation of [3H]dThd into trichloroacetic acid-insoluble material. (a) Infection of WC+ bacteria; (b) infection of recA- bacteria; (c) infection of recA -B- baoteria. The data are expressed as percentageof maximum incorporation, which in all oases was taken from the wild type results at 30 min. Host DNA synthesis in recA- and recA- B- cells was inhibited by pretreatment with u.v. as described in Materials and Methods. The amount of Xspecific radioactivity incorporated in infected W.C+ cells was determined by DNA hybridization as described by Skalka & Hanson (1972). (A) Infected with wild type; (0) infected with red- ; (a) infected with garn- ; ( a) infected with red -gum- ; (0) uninfected cells. In the recA - B infection non-supressible red*,, and ga7ns mutants were used.

192

L. W.

ENQCJIST

AND

A. SKALKA

to ten minutes after infection of the recA - host. The results (not included here) showed no significant differences between wild type, red-, gam-, and red-gamphages up to eight minutes after infection. Thus, we conclude that the defects are probably specific for late times. Biological data, which incorporate results from the experiments described in Figure 4, are summarized in Table 2. As expected from results of the rate measurements, the yield of gum- phages was as high as the wild type in recA-Bcells. The yield of red - and red-gum- phage was low but equal to that for red- phages in the recA or ret+ hosts. Our results with the recA-Bhost verify the findings of others, who showed that the burst size of red-gam- mutants in these cells was significantly lower than for wild type phage (Sironi et al., 1971). Manly et al. (1969) and Zissler et al. (1971) have suggested, on the basis of plaque morphology, that red-gum- phage multiply normally in recA-Bhosts. We conclude that, in this case, plaque morphology reflects some other parameter of phage growth. TABLE 2 Growth of h mutants after infection Infection

Infective

of ret + and ret-

centers.1

hosts

Yield$

ret+ hod A wild type red113 gam5 red113 gam5

1.00 0.82 0.84 0.88

1.00 (167) 0.34 0.37 0.16

ret A- host h wild type red270 gam210 red270 gum210

1.00 0.67 0.71 0.57

1.00 (60) 0.40 0.36 0.09

ret A-Bhost X wild type Ted113 gum5 red113 gam5

1.00 1.16 0.88 0.81

1.00 (90) 0.38 1.07 0.30

Conditions for infection are described in the legend for Fig. 4. The numbers are averages of results from 2 experiments for ret + infections, 7 experiments for re.cA-, and 3 experiments for ret A -B- infections. t Determined between 5 and 10 min after infection, by plating a suitable dilution on E. coli C600. Results are normalized to wild type values. 2 Yield per infected cell normalized to wild type results, which e,re shown in parentheses.

Our results show very little difference in the number of infective centers for red-gum- as compared to red- or gum- (or wild type) infected recA - cells. This is in contrast to the data of Zissler et al. (1971), which indicate that red-gum- mutants are only 10 to 20% as effective as wild type or the single mutant phage in producing infective centers in a recA- host. This discrepancy in the two sets of data may reflect bacterial strain differences; otherwise the recA- and ret+ data agree quite well. (d) Tests for breakdown of phage and host DNA in recA- bacteria The recA- strains characteristically have a high rate of spontaneous and WV.induced breakdown of DNA (Howard-Flanders & Theriot, 1966; Clark et al., 1966).

h REPLICATION

193

This breakdown is apparently due to a nuclease determined by the recB and recC cistrons acting concertedly (Buttin & Wright, 1968: Oishi, 1969; Barbour & Clark, 1970). The data shown in Figure 5(a), (b), (c) and (d) are from experiments designed to determine if the defect in DNA synthesis and growth of Ted-, yarn- and red-gamphages was due to destruction of intracellular phage DNA. DN.4 was pulse-labeled at early (Fig. 5(a)), intermediate (Fig. 5(b)) and late (Fig. 5(c)) times and then chased with excess unlabeled thymine. Breakdown of the DNA made during the labeling period was followed by measuring trichloroacetic acid-insoluble counts during the chase. To examine stability of DNA long after lysis would have normally occurred, we constructed phages with an additional mutation in a lysis function (gene X) I I i (a) Phage DNA -Label

I

1

1

1

I 1

Tune offer infectlon(m~n)

5. Stability of phage and host DNA made during infection of reck bacteria. (A) Infected with wild type; (0) infected with red- ; ( l ) infected with garn- ; (a) infected with red-gam-. Phage DNA was la.beled with [3H]thymine (methyl labeled, 11.7 Ci/mmol) during the following intervals after infection: 0 to 15 min (a); 13 to 15 min (b); 23 to 25 min (0) and (d). Host DNA was pre-labeled with d[r4C]Thd (methyl labeled, 65*3mCi/mmol; New England Nuclear) as described in Materials and Methods. The infected cells were suspended in 5 ml minimal medium (plus 5 pg thym.ine/ml) at 47°C for 5 min with aeration, followed by aeration and incubation at 37°C for the duration of the experiment. At the indicated time, enough [3H]thymine was added to give a spec. act. of 10 cLCi/pg thymine. At the end of the labeling period, 5 ml of warmed medium oonmining 2 mg unlabeled thymine/ml were added as a chase solution. The stability of pre-labeled host DNA and phage DNA made during the labeling period was determined by measuring the trichloroacetic acid-insoluble radioactivity (described in Materials and Methods) at the indicated time intervals after the chase. The experiment described in (c) and (d) was conducted using phage carrying the lysis-defective S mutation in addition to the red- and gem- mutations. (d) shows the stability of host DNA during the 4 infections as measured simultaneously with the stability of the phage DNA made during a late pulse (c). FIG.

194

L.

W. ENQUIST

AND

A. SKALKR

(Fig. 5(c) and (d)). The results show that the phage DNA remained trichloroacetic acid-precipitable regardless of when it had been labeled. Host DNA stability was measured simultaneously in all experiments by prelabeling the cells with d[lV]Thd. No solubilization of host DNA was observed after infection with the wild type or ret- phages. Some breakdown was observed in the gam-Sinfection but, as shown in Figure 5(d), after 30 minutes the greatest amount of DNA breakdown was found in the cells infected with red-gam-S- phage. By 80 minutes, 40% of the prelabeled host DNA had been rendered acid-soluble. As mentioned above, a similar breakdown of bacterial DNA has been shown to occur in uninfected u.v.treated bacteria. Another, more sensitive, measurement of host DNA breakdown was made using density-shift techniques (Skalka et al., 1972). In these tests (data not shown) host DNA was density-labeled with Da0 and 15N, and phage infection was carried out in light medium containing d[3H]Thd. Both wild type and red- DNA extracted at late times had the expected light density, while gam- and red-gamDNA exhibited between 20 and 25% substitution of heavy isotope from the host, as judged from its intermediate density in CsCl. Thus, it is clear that estimates of DNA synthesis at late times (based on incorporation of externally added [3H]thymine or thymidine) in the gam- and red-gaminfection of recA cells oould be low by as much as 20 to 25%. (e) Sedimentation and density analyses of intracellular phage DNA by wild type, red-, gam- a& red-gam- phages

produced

(i) recA- Bacteria Intracellular phage DNA was labeled with dr3H]Thd between the 23rd and 25th minutes after infection (pulse) ; at the 25th minute, excess unlabeled dThd was added (chase). DNA was extracted at 25, 35 and 45 minutes as described in Materials and Methods. To insure quantitative recovery of late intermediates, DNA maturation (formation of linear monomers with cohesive ends) was prevented by inclusion of an A head gene mutation (Weissbach et al., 1968; Skalka, 1971a). The intracellular DNA obtained was analyzed by sedimentation in neutral and alkaline sucrose gradients. Analyses of chase DNA isolated at 35 minutes are shown in Figure 6. Results from the A- infection (Fig. 6(a) and (d)) were almost identical with those already published (Skalka, 197la; Skalka et al., 1972), in which the late intracellular X DNA of wild type and red- mutants was observed to contain no ciroular forms, but consisted mainly of linear monomers and concatemers. Results from neutral sucrose analysis of A-red-gamDNA (Fig. 6(c)) were markedly different. DNA in this preparation was mainly in the form of circular monomers with approximately half of these covalently closed and half relaxed. DNA in the A-gampreparation (Fig. 6(b)) looked almost like a mixture of the A- and A-red-gamDNAs. Much of it sedimented at the position of covalently closed and relaxed circles, but there was also a distinct peak (containing about 25% of the total mass) at the same position as the main peak of A- DNA. In alkaline sucrose, the differences were equally striking. The A- DNA (Fig. 6(d)) sedimented in a broad band composed of molecules apparently much longer than DNA (Fig. 6(f)) sedimented as two compomature single chains. The A-red-gamnents; approximately half of the DNA was in the dense pad (as expected of covalently

X REPLICATION

7

(f) A A-red-gums

(cl A A -red -gm-

Fraction no

FIG. 6. Sedimentation analysis of late intracellular phage DNA from infected recA- bacteria. Gradients (a) to (c) are neutral sucrose and (d) to (f) are alkaline sucrose. Infected cells (multiplicity of infection, 2) were suspended in 5 ml minimal medium (plus 5 pg thymine/ml) at 47°C for 5 min with aeration, followed by aeration and incubation at 37°C for the duration of the experiment. At the 23rd min, enough d13H]Thd (methyl labeled, 20 Ci/mmol; New England Nuclear) was added to give a spec. act. of 20 &X/O.25 pg dThd in the presence of 5 pg unlabeled thymine/ ml. At the 25th min enough unlabeled dThd was added to give 2 mg/ml final concentration (chase). DNA was isolated at 25, 35 and 45 min as described in Materials and Methods. Only the results from the 35mm chase are shown here. Neutral and alkaline sucrose gradients (5 ml) were prepared and the samples applied as described by Skalka et al. (1972). All gradients contained as a marker 32P-labeled linear monomer X DNA extracted from phage particles (broken lines). In neutral gradients, arrows indicate the position of the linear marker (L), relaxed circular molecules (C) and covalently closed circular molecules (CC). In alkaline gradients, arrows mark the position of the single-stranded linear marker (L), single-stranded circles (C) and covalently closed circles (CC). All gradients contain dense pads consisting of 0.2 ml 70% sucrose and 0.2 ml 80% sodium iothalmate (Angio-Conray, Mallinckrodt Pharmaceuticals) which had been placed on the bottom of the tubes. Fractions containing this material are indicated by black rectangles. Sucrose gradients were centrifuged in the SW50.1 rotor at 28,000 revs/min for 210 min at 11°C (neutral gradients) and 45,000 revs/min for 120 min at 11°C (alkaline gradients). Sedimentation is from right to left. Neutral gradients contained about 0.4 pg bacterial DNA (unlabeled) and about 0.02 to 0.06 pg phage DNA (spec. act. from 1 to 5 x 105 cts/min/pg). Alkaline gradients contained about 0.8 pg bacterial DNA and 0.04 to 0.12 pg phage DNA.

closed circles under these conditions) and the other half slightly ahead of the mature single chain “marker “. Longer centrifugation (not shown) revealed that this second component consisted of two types of molecules in approximately equal amounts; one which sedimented with the linear marker and the other as expected of singlestranded monomer circles (see also Fig. S(h)). Again, the A-gamDNA looked like a mixture of the other two preparations (Fig. G(e)), with a significant fraction of the

196

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total sedimenting as single-stranded polynucleotide chains of longer than monomer length. These faster sedimenting molecules seemed to accumulate in two small peaks; our calculations indicate that dimer circles would be found among those molecules in the slower peak (fractions 18 to 20) and that trimer circles would be among those in the faster peak (fractions 15 to 16). However, this coincidence may be fortuitous and must be checked by other methods. A summary of the results from neutral sucrose sedimentation of DNA from the pulse and chase experiments is shown in Figure 7. The percentage of labeled DNA in four main species was estimated from the amount of radioactivity in the characteristic regions of the neutral sucrose gradients (“shelf”, 56 to 75 S, covalently closed, and “relaxed ” circle regions). I

I

(b) X A-gum-

I

I

I

I

Covolentiy closed

Covolently closed

i

Ci&S

OS’ 0

20

k-’

’ 30

Chase ’ ’ 40

’

:

0

20

30

40

0

20

30

40

Time after infection (mtn)

FIG. 7. Fate of various intracellular forms of maturation defective A- (a), A-gam(b) and A -red-gum(c) DNA during infection of TecA - bacteria. The data were obtained from the experiment described in the legend to Fig. 6. The percentage of 3H radioactivity in 4 distinct species from neutral gradients was calculated as follows (see for example, gradients in Fig. 6) : the species termed “&eEf” were fractions 1 t.o 8 (A); 56 to 75 S, fractions 8 to 16 f A); covalently closed circles, fractions 17 to 20 (a); relaxed circles, fractions 22 to 24 ( n ). In all 3 DNA preparations, 10 to 15% of the radioactivity sedimented more slowly than linear monomers and is not included in this Figure.

The major fraction of the pulse-labeled DNA from all three preparations was found in the dense shelf. In the chase, most of the label appeared in the 56 to 75 S fraction in the A- infection (Fig. 7(a)) but in covalently closed circles in the A-gam(Fig. 7(b)) and A-red-gum(Fig. 7(c)) infections. The amount of radioactivity in relaxed circles seemed to increase with time after A-gamand A-red-gaminfections. An increased amount of radioactivity was found in the 56 to 75 S region after the chase in the A-gaminfection, while a decreased amount of radioactivity in this same region was observed after the chase with A -red -gam- _The data obtained from the A-gamand A-red-gampulse and chase experiment are very similar to those found by Carter et al. (1969) to be typical of replication at early times, while the

197

X REPLICATION

TABLE 3 closed circular molecules by red- and red-garaat various times after infection of u.v.-treated recA- cells

Synthesis of covalently

Time after infection (min) Pulse 5-10 Chase lo-15 Pulse 10-15 Chase 15-20 Pulse 23-26 Chase 26-31

red Cts/min in relaxed DNA

mutants

red- gamCts/min in relaxed DNA1

% CDCp

519

2607

16.6

0

5461

0

1298

510s

20.3

175

9058

1.8

1085

3198

25.3

189

4218

4.3

2439

5306

30.7

260

11,060

2.3

1367

2865

32.3

0

5316

3353

6449

34.2

238

13,365

Cts/min in ccc?

Cts/min in ccc

% ccc

0 1.7

t Counts per min found in band corresponding to covalently closed circular DNA. coo, covalently closed circular molecules. $ Counts per min found in main band. $ Expressed as O/e of total counts recovered in the covalently closed circle and main band. Recovery from the gradient was better than 85%. Cells were grown, treated with U.V. and infected at a multiplicity of infection of 1.5 as described in Materials and Methods. At the indicated time, 5 ml of the culture were added to warmed medium containing 5 pg thymine/ml and 250 pCi d[3H]Thd (methyl la$beled, 11.0 Ci/mmol; New England Nuclear). At the end of the labeling period (pulse), excess unlabeled dThd (2 mg/ml) was added and 2 ml of the culture were immediately taken for DNA extraction (see Materials and Methods). After 5 min growth in the presence of excess unlabeled dThd, 2 ml were taken for DNA extraction (chase). The extracted, dialyzed DNA preparations were then analyzed in ethidium bromide/ CsCl gradients as described in Materials and Methods. Total trichloroacetio acid-precipitable radioactivity in each gradient was from 1 to 2 x lo4 cts/min. The amounts (in pg) of phage and bacterial DNA are similar to those given in the legend for Fig. 6.

A - results correlated well with the observations of Skalka (1971a), Carter & Smith (1970) and Weissbach et al. (1968) for X DNA replication at late times. Carter & Smith (1970) reported that synthesis of covalently closed circles stopped after 15 minutes in a normal h infection (multiplicity of infection, 15) of wild type bacteria. By using pulse and chase experiments, as described for Figures 6 and 7, we were able to show (Table 3) that covalently closed circular DNA molecules, identified by their density in ethidium bromide-CsCl gradients, were made abundantly throughout the infectious cycle of red-gum- (A+) mutants in u.v.-treated recAbacteria. The rate of synthesis of covalently closed circular molecules seemed to increase during infection. Late after infection (23 to 26 minutes) as much as 32% of the pulse-label was found in covalently closed molecules; at no time was more than 4% of the red- pulse-labeled DNA found in this form. Control experiments proved that covalently closed circle formation was not dependent on U.V. irradiation, because red-gam-, but not red- or wild type phage, produced a significant proportion of these molecules late after infection of untreated recA- bacteria. To verify the observations made in the pulse-chase experiments with maturation defective phage, we analyzed the intracellular DNA from u.v.-treated recA- cultures infected with A + phage. In this case the isotope was present from the onset of infec-

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16 12 8 4 C

0

IO

20

30 Fraction no.

and density anctlysis of intracellular phsge DNA from infected recA FIG. 8. Sediment&ion baoterie. Gradients (a) to (d) are neutral sucrose; (e) to (h), alkaline sucrose; ctnd (i) to (1) are ethidium bromide/CsCl density analyses. DNA W&S labeled from 0 to 25 min with d[sH]Thd (methyl labeled, 20 Ci/mmol; New England Nuclear). Specific activity of d[eH]Thd was 10 pCi/ 0.12 pg dThd in the presence of 5 pg unlabeled thymine/ml. DNA was extracted at 25 min as described in Materials and Methods. Methods and symbols are described in the legend to Fig. 6, with the following exoeptions: alkaline sucrose gradients were centrifuged at 45,000 revs/min for 120 min at 11°C in the SW50.1 rotor; the speo. act. of the DNA was between 1 to 3x lo5 cts/min/pg; in ethidium bromide/CsCl density gradients, mrows mark the density of super-coiled molecules (S); ethidimn bromide/CsCl gradients contained about 4 pg baoteria.1 DNA and 0.2 to 0.6 pg phage DNA.

tion. It should be emphasized that the continued presence of isotope during the entire lytic cycle insures that all stable intermediates oharaoteristic of both early and late times will be labeled. Even so, results from neutral sucrose analysis confirmed that both wild type (Fig. 8(a)) and red- intracellular DNA (Fig. S(b)) consisted mainly of monomeric linear molecules and concatemers, although with red- there seemed to be less DNA at the concatemer positions. Both gum- (Fig. 8(c)) and red-gamDNA (Fig. 8(d)) contained little or no monomeric linear DNA (fractions 21 to 23), but a large proportion of relaxed (fractions 19 to 21) and covalently closed circles (fractions 13 to 17). Estimation of the percentages of concatemers from the wild type and red- alkaline gradients (Fig. 8(e) and (f)) is complicated by the fact that many molecules in this form sediment at the same rate as single-stranded circular DNA (see also Skalka et al., 1972). However, comparison of results in Figure 8(g) and (h) suggests that little of the DNA in the gam- infection and less from the red-gam- infection accumulates in concatemers. Identification of both relaxed circular and supercoiled molecules has been

h REPLICATION

199

verified by electron microscopy of DNA samples purified from sucrose gradients. Approximately 35 to 45% of the radioactivity in alkaline sucrose gradients of gamand red-game intracellular DNA sedimented as sharp peaks of circular and linear monomers, with slightly more present in the linear form. This result suggests that most of the relaxed circular molecules observed in neutral sucrose gradients contained an interruption in only one strand. The results from ethidium bromide/CsCl banding of DNA from these preparations verified that less than 5% of the wild type and redDNA (Fig. 8(i) and (j)), but about 25% of the gam- and red-gam- DNA (Fig. S(k) and (l)), was in the form of supercoils. Results obtained with substitution h pbioll (red-g&m-) and h pbio72 (red-) were similar to those already shown for the red - and red -game point mutants and therefore are not presented. (ii) ret+ Bacteria It is clear from the measurement of the rate of DNA synthesis in ret+ bacteria that both red - and gam- mutants are defective when compared to wild type phages (Fig. 4(a)). These defects are, in fact, indistinguishable from those observed for the recA - bacteria. Our analysis of DNA preparations from infected ret+ bacteria showed further similarities. In ret+ bacteria, host DNA synthesis is not easily shut off with low doses of U.V. Therefore, in order to eliminate ambiguities due to bacterial DNA synthesis, intracellular DNA was analyzed before and after purification of the phage species by density-shift techniques. To this end, host DNA was density-labeled with D,O and 15N (and a 14Cradioactive label) and phage infection was carried out in light medium. Newly synthesized DNA was labeled with d[3H]Thd between the 25th and 27th minutes after infection. At the 27th minute, excess unlabeled dThd was added (chase) and that DNA was extracted at the 35th minute. Light (phage) DNA was purified as described by Skalka et al. (1972). To insure quantitative recovery of late intermediates, the A head gene mutation was again included as described in section (e) (i) above. Results from neutral sucrose sedimentation analysis of unfractionated DNA and neutral and alkaline analysis of purified intracellular phage DNA are shown in Figure 9. As judged by DNA-DNA hybridization tests, from 10 to 20% of the unfractionated 3H-labeled DNA was of bacterial origin. Almost all of the 14C-labeled (and presumably the 3H-labeled) E. coli DNA in the unfractionated preparations sedimented in neutral sucrose at 55 to 75 S. The purified phage DNA contained less than 1y. bacterial DNA contamination, as judged by l*C content and hybridization specificity. The neutral gradients (Fig. 9(e) and (h)) show that these preparations contained few fast-sedimenting molecules, as compared to the unfractionated DNA (Fig. 9(a) and (d)), presumably (as has been discussed before by Skalka et al., 1972) because such molecules are selectively sensitive to the purification procedures. As with the recA- data, results with unfractionated and purified A- (Fig. 9(a), (e) and (i)) and A-redDNA (Fig. 9(b), (f) and (g)) were almost identical to those already published (Skalka et al., 1972), and indicated that intracellular h DNA consisted mainly of linear monomers and concatemers. Data obtained from sedimentation analysis of A-gamand A-red-gamDNA were decidedly different. Even in the unfractionated preparations (Fig. 9(c) and (d)), distinct peaks of radioactivity were found at positions of covalently closed and relaxed circles. The presence of covalently closed circles was also verified by density

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IO 20 Fraction no.

FIG. 9. Sedimentation analysis of late intracellular phage DNA from infected ret+ bacteria. Symbols are as described in the legend to Fig. 6. Gradients (a) to (d) are neutral suorose analyses on unfractionated samples; (e) to (h) are similar analyses on purified DNA preparations; (i) to (1) are alkaline sucrose analyses of purified DNA preparations. Methods for density-shift purification of phage DNA have been described by Skalka et al. (1972). Density-labeled cells were infected with the appropriate amber mutants at a multiplicity of infection of 2. Infected cells were suspended in 4 ml light minimal medium (plus 10 pg thymine/ml) at 47°C for 5 min with aeration, followed by aeration and incubation at 37°C for the duration of the experiment. At the 25th mm, d[3H]Thd (methyl labeled, 20 Ci/mmol; New England Nuclear) was added to give 25 pCi/ml. At the 27th min unlabeled dThd was added to a final concentration of 2 mg/ml (chase). DNA was isolated at 35 min as described in Materials and IMethods. Neutral and alkaline sucrose gradients were prepared and run as described in the legend to Fig. 6. Neutral gradients (unfractionated DNA) contained about 0.4 pg bacterial DNA (YXabeled) and about 0.02 to 0.05 pg phage DNA (spec. act. about 1 x lo6 cts/min/pg). Neutral and alkaline gradients of purified DNA contained about 0.02 pg phage DNA.

analysis in ethidium bromide/&Cl and sedimentation in alkaline sucrose (data not included). The neutral sucrose data (Pig. 9(g) and (h)) show that both the A-gamand A-red-gampurified preparatiorrs contained predominantly covalently closed and relaxed circular molecules. The alkaline sucrose results (Fig. 9(k) and (1)) suggest that a small proportion of this DNA is also in the form of concatemers. All of the results obtained with the red-gam-ADNA are strikingly similar to those for ganz-ADNA from recA- cells (see Fig. 6(b) and (e)). The significance of this similarity will be discussed later. (iii) recA-B-

Bacteria

Preliminary tests of recombination frequencies indicated that our recA -B- strain contained a weak (undefined) suppressor, Therefore, in these experiments non-suppres-

h. REPLICATION

201

sible red- and gam- mutants were used. In control experiments, with ret+ and recAbacteria, these mutants gave results comparable to the red and gam ambers. In order to minimize ambiguities caused by maturation, since our A mutant was an amber, DNA was labeled and extracted at a slightly earlier time (20 to 22 min and 25 min, respectively). Results with wild type DNA (Fig. 10(a)) indicate that the preparations so obtained had the sedimentation properties typical for late times and (as judged by the small amount of intracellular wild type DNA, which sediments with the phage DNA marker), very little maturation had taken place.

-0

-Density

Frcction no.

FIG. 10. Sedimentation and density analysis of intracellular phage DNA from infected recA -Bbacteria. Gradients (a) to (d) are neutral sucrose; (e) to (h) alkaline sucrose; and (i) to (I), ethidium bromide/C&l density rtnalyses. Methods and symbols are described in the legend to Fig. 6 with the following exceptions: at the 20th min after infection, enough d[3H]Thd (methyl labeled, 20 Ci/mmol; New England Nuclear) was added to give final spec. act. of 50 nCi/pg dThd. At the 22nd min unlabeled dThd was added to a final concentration of 2 mg/ml (chase). DNA was isolated at the 25th min as described in Materials and Methods. Neutral and alkaline sucrose gradients contained about 0.8 pg unhabeled bacterial DNA and 0.05 to 0.1 pg phage DNA. Ethidium bromide/ CsCl gradients contained about 2 pg unlabeled bacterial DNA and from O-1 to O-3 pg phage DNA. The specific activity of the phage DNA was 2 to 3 x 10s cts/mi.n/pg. In ethidium bromide/C&l gradients, arrows mark the density of supercoiled molecules (S).

In contrast to results obtained from similar experiments with WC+ or recAbacteria (Figs 6 and 9), the intracellular DNA from the wild type (Fig. 10(a), (e) and (i)) and gam- mutant infections (Fig. 10(c), (g) and (k)) of the weA-Bhost were indistinguishable. The red- (Fig. IO(b), (f) and (j)) and in this case also the red-game DNA (Fig. IO(d), (h) and (1)) showed sedimentation behavior almost identical to 14

202

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red- DNA from ret+ and recA- bacteria. As mentioned above (section (e) (i)), when compared to the wild type DNA all of the red- preparations contained less material that sedimented at the concatemer positions and more that sedimented as linear monomers. We conclude that, although quantitative differences exist, inrecA - Bcells all of the phage mutants tested produce linear structures which seem normal for late times after infection. Quantitative differences observed between wild type and red- DNA in this as well as in the ret + and recA - hosts might be expected if the length of concatemers somehow reflects the rate (efficiency) of DNL4 synthesis. (f) Analysis

of the nature and location of the break in intracellular circular DNA from recA- cells

gam-

and red-gam-

(i) Hybridization of single-stranded circles with puri$ed r and 1 strands of X As noted above (section (e) (i)), results from alkaline sucrose sedimentation analysis of red-gam- DNA produced in recA - cells indicated that most relaxed circular molecules contained an interruption in only one strand. The results obtained from hybridization experiments (Table 4) show that both the purified single-stranded circular and linear molecules anneal to 1 and r single strands with efficiency equal to unfractionated 32P-labeled phage DNA included in the reaction mixture. The sum of efficiency with both strands is equal to the percentage expected for unfractionated DNA. We conclude, therefore, that breaks occurred in either strand. Because breakage of covalently closed circles by radioactive decay, thermal stress or nuclease activity will raise the “noise” level of randomly derived single-stranded circles, a small bias towards one strand or the other would probably not be detected in this experiment. TABLE 4

of red-gam- single-stranded circular and linear DNA puri$ed from an alkaline sucrose gradient to separated 1 and r strands of h DNA

Annealing

DNA

Hybridization 1 strand T strand (%I (%I

Circles; sH-labeled Phage DNA; 32P-labeled

31 25

38 34

Linears; 3H-labeled Phage DNA; s2P-labeled

23 20

29 27

Input radioactivity (either 3H or s2P) was between 3 to 5 x 10s cts/min in less than 0.5 pg DNA. The recA- cells were grown, treated with U.V. and infected with red-gamphage at a multiplicity of infection of 1. Infected cells were suspended at zero time in 4 ml medium at 46°C oontaining 5 pg thymine/ml and 100 PCi d[sH]Thd (methyl labeled, 12 Ci/mmol; New England Nuclear). After 5 min at 46”C, the infected cells were transferred to 37°C for 20 min. DNA was then extracted as described in Materials and Methods. One ml of the DNA preparation (1.8 x lo5 cts/min/ml; 1 pg phage DNA and about 6 pg unlabeled bacterial DNA) was layered on a 26-ml 5 to 20% alkaline sucrose gradient with a 2-ml bottom pad of 70% sucrose plus 80% sodium iothalmate in a 1: 1 ratio. Centrifugation was for 230 min at 26,000 revs/min and 1l’C in the SW27 rotor. The DNA in fractions containing single-stranded circles and linear monomers was precipitated with ice-cold trichloroacetic acid using T4 DNA and bovine serum albumin as carrier. DNA hybridization was done as described by Skalka (1971a) and Skalka & Hanson (1972). Purified T and I strand (3 pg immobilized DNA per filter) were obtained from Dr Waolaw Szybalski.

A REPLICATION

203

(ii) Structure d the lesion Most of the known E. coli nucleases capable of producing interruptions in DNA generate molecules with 5’-phosphate and 3’-hydroxyl ends. We tested different preparations of intracellular phage DNA for the presence of nicked or gapped circular molecules with such interruptions using a combination of T4 polynucleotide ligase and T4 DNA polymerase (Masamune et al., 1971; Weiss et al., 1968; Becker et al., 1967). In our tests (Table 5), the assay for enzyme activity was conversion of hydrogenbonded circles to covalently closed circular molecules, as determined by alkaline sucrose sedimentation (see Materials and Methods). All reaction mixtures contained 32P-labeled DNA enriched for hydrogen-bonded circles (60 to 70% circles) as an internal control. In each mixture tested, the T4 ligase alone converted 13 to 14% of the 32P-labeled DNB to covalently closed circular molecules. The combination of T4 ligase and T4 DNA polymerase increased the efliciency of closure to 20%. These results are in excellent agreement with those of Masamune et al. (1971), who used the T4 enzymes and similarily prepared hydrogen-bonded circles. We observed no increase in the proportion of covalently closed circles when intracellular DNA from wild type or red - infections was tested with the ligase or ligase plus polymerase. This was expected because few relaxed circular molecules were observed in these preparations. However, in the gam- infections where relaxed circular molecules are found in abundance, there was also little increase in the proportion of covalently closed circular molecules. Control experiments showed that the addition of four units/ml of TABLE

Treatment

of DNA

5

extracted from infected cells with T4 polynucleotide and polymerase in vitro

ligase

Percentage

Phage

Enzyme

Wild type

None Ligase Ligase + polymerase

red -

None Ligase Ligase + polymerase

pm -

None Ligase Ligase + pblymerase

red-pm

-

None Ligase Ligase + polymerase

sedimenting as covalentlyt closed circles 3H-labeled s2P-labeled intracellular DNA DNA (control)j: 5.7 5.8 6.9 5.2 4.1 6.3 15.3 15.1 17.7 16.7 17.4 18.9

0.0 13.5 20.3 1.1 12.9 19.1 0.03 14.2 21.6 0.05 14.4 19.8

The TecA- cells were grown, treated with U.V. and infected at a multiplicity of infection of 1. Infected cells were suspended at zero time in 2.5 ml medium (46Y) containing 5 pg thymine/ml and 100 &i d[sH]Thd (methyl labeled, 12 Ci/mmol; New England Nuclear). After 5 min, the infected cultures were transferred to 37°C and the DNA extracted at 25 min after infection. Enzyme treatment and other techniques are described in Materials and Methods. Each gradient contained from 1 to 2 x lo4 ots/min in 3H and 1 x lo4 cts/min in 32P radioactivity. $ Calculated as a percentage of the total input radioactivity (cts/min). $ 3aP-labeled DNA contained 60 to 70% hydrogen-bonded circles and 30 to 40% linear molecules as judged by neutral sucrose gradient sedimentation.

204

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SKALKA

polynucleotide kinase to ligase or to ligase and polymerase had no effect on the sealing efficiency. Thus, it seems unlikely that the laok of closure was due to the absence of a 5’-phosphate at the site of the interruption. These data indicate that the interruptions present in the relaxed circular molecules formed in the gam- and red-gaminfections of recA - bacteria do not contain nicks or gaps similar to those presumed to be present in the hydrogen-bonded ciroles. (g) X-Specific RNA synthesis in recA- bacteria infected with wild type, red-, gam- and red-gam- phages As noted in section (b) above, the DNA produced by red-gam- phage during infection of recA- bacteria is not efficiently packaged into infectious phage particles. This defect in morphogenesis could result from a direct block due, perhaps, to the abnormal TABLE 6 Analysis of )I messenger RNA plproducedin recA- bacteria infected with wild type, red-, gam- and red-gam- phages Total RNA &/ml ( x 107)

Wild type

2-4 6-8 20-22 28-30

5.6 5.2 5.1 5.3

1.87 1.37 8.57 11.32

89.6 74.7 39.9 26.3

10.4 25.3 61.1 73.7

red-

2-4 6-8 20-22 28-30

3.4 4.1 4.8 46

1.86 3.92 14.16 12.97

83.6 78.3 31.7 22.4

16.4 21.7 68.3 77.6

gam -

2-4 68 29-22 28-30 2-4 6-8 20-22 28-30

5.2 6.8 6-O 5.0 4.8 5.5 6.8 5.5

1459 3.16 10.42 9-36 2.94 2.16 8.29 9.77

83.4 64.8 25.8 18.9 77.2 72.4 28.0 19.5

16.6 35.2 74.2 81.1 22.8 27.6 72.0 80.5

6-8

1.9

0.05

80.2

19.8

Tt?d

- gam -

Uninfected

Lamb&t-specific RNA (%I

Distribution of X hybridizable counts$ Right half Left half (%)

Time of pulse W4

Phage

Cells were grown and infected at a multiplicity of infection of 1 as described in Materials and Methods. No u.v. treatment was used. Infected cells were suspended in 32 ml medium (46’C) containing 10 pg thymine/ml at zero time. After 5 min, the cultures were transferred to 37“C for the durrttion of the experiment. At the indioated intervals, S-ml samples were transferred to tubes containing 0.4 ml [3H]uridine (methyl labeled, 26 Ci/mmol; New England Nuclear). After 2 min the cultures were poured over frozen medium containing 20 mM-sodium azide. RNA was extracted according to the method of Bevre & Szybalski (1971). Determination of the distribution of h-specitic RNA was accomplished in 2 steps. First, total pulse-labeled RNA was hybridized to X DNA illters. The hybridized RNA was then eluted and rehybridized to filters containing purified right and left halves of X DNA. Right-half and left-half Eambda DNA molecules were isolated as described by Skalka (1971b). t Hybridization conditions were as described by Bevre & Szybalski (1971). The mixture oontamed less than 1.6 pg total (bacterial+X) RNA. The filters oontained 6 pg denatured h DNA. Blanks (percentage binding to filter with no DNA) of 0.20% have been subtracted. $ Input radioactivity was 1500 to 2000 ots/min. The average amount bound to both filters was 64.7%.

205

h REPLICATION

structure of the intracellular DNA. On the other hand, the block could be indirect; for example, the abnormal structures might be poor substrates for late mRNA synthesis. We analyzed the mRNA produced in all four mutant infections to distinguish between the two alternatives. The results (Table 6) show no significant differences in either the rate or temporal control of transcription in the four infections. In all oases, the rate of transcription was low at early times and mainly right-arm genes were transcribed. At late times, the rate was higher and mainly left-arm genes were transcribed. Because left-arm mRNA (and presumably head and tail proteins) is synthesized normally in the red-gam- infection, we conclude that the block is probably direct, that is, red-gam- (circular) DNA is structurally unsuitable for packaging. This conclusion is supported by preliminary evidence from electron microscopy of thin sections, which indicates that empty head structures accumulate in recA- bacteria infected with the red-gum- mutant.

4. Discussion (a) Function of gam gene Results from our analysis of the molecular structure of gam- and red-gum intracellular DNA provide important clues to at least one aspect of gam gene function. E‘igure 11 illustrates the program of replication for a typical wild type infection, in which X DNA enters the cell and rapidly circularizes and replicates once or twice to form daughter circles (Schnos & Inman, 1970; Tomizawa & Ogawa, 1968). Thereafter, a second mode of replication ensues, the product of which is concatemers and not circular molecules (Skalka et al., 1972; Skalka, 1971a; Kiger & Sinsheimer, 1970). It is clear that the gam mutation strikingly alters this normal program of X replication in wild type and recA - cells (see Table 7 for summary). In such mutant infections, monomeric circular molecules are produced continuously throughout the infectious cycle. At late times after infection, as exemplified by results in recA- cells (Pig. 7 ;

7

TABLE

iSummary of results from analyses on the rate of synthesis and structure of late phage DNA from various combinations of mutant infections ret+ Bacteria

Phages : Wild type

Late rate

Late structures

Late rehe

recA Late structures

recA- BLate Late rate structures

N

N

N

N

N

N

Ted-

D

N’

D

N’

D

N’

gam -

D

Circles (and some concstemers)

D

Circles (and. some) concatemers)

N

N

red- gam-

D

Circles (snd some conoatemers)

D

Circles (no concatemers)

D

N

Abbreviations used: N, normal; late structures are typically linear monomers and concatemers; N’, mostly normal structures, but some quantitative differences when compared with wild type; 13, defective; circles, circular monomers, both covalently closed and “nicked or gapped”.

206

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Replication “early”stoge

BC nucleose-sensltive

FIG 11. A schematio representation of the infectious cycle of bacteriophage Ear&da. The model emphasizes a replication program consisting of distinct early and late stages, demonstrates a possible role for recombination function in phage development and stresses the essential nature of concatemers in phage morphogenesis.

Table 3), the labeling pattern of gam-

DNA seems typical of early replication (Carter

et al., 1969). The simplest conclusion is that the gam gene produot is required for the

transition from the early (circle producing) to late (concatemer producing) mode of replication. Infection of the recA- host with a gam mutant containing a defect in the red function (fee- infection) results in a similar alteration of the replication program, except in this case, monomeric circular molecules are made almost exclusively. We conclude, therefore, that those few concatemers which arise in the presence of red (ox in the presence of ret in the ret + host) are formed through the action of these recombination proteins. Comparison between amounts of intracellular DNA and plaque-forming phages produced in recA- infections (Fig. 3) shows that red-gamDNA, in contrast to gam- DNA, is not efficiently matured and packaged. Thus, we assume that in the gam- infection, phage production is also dependent on some function of the recombination system. We will discuss the relation between phage formation and ooncatemer production by the red and ret systems in section (b) below. The requirement for the gam product in the transition from early to late replication is not observed in the absence of the host’s reel3 gene product. In the recA-B-

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host, gam DNA is synthesized at a normal rate and no intracellular circular forms are detected at late times (Table 7). This suggests that the gam product plays a negative role in replication, perhaps as an antagonist of the recB product (Zissler et al., 1971; Sironi et al., 1971). Unger et al. (1972) and Unger & Clark (1972) have recently reported evidence which suggests that the gam protein can inhibit BC nuclease action in vitro. Our own results (Fig. 5(d)) provide direct evidence that the gam gene product can inhibit the presumed action of the BC nuclease. These data also show that the red gene products can repair some of the damage that occurs in the absence of gam; this property of red will also be discussed in section (b) below. It had been suggested that the failure of red-gam- phages to grow in recA - hosts might be due to nucleolytic degradation of incoming phage DNA (DeLafonteyne, 1971; Lindahl et al., 1970). Our data do not support this notion. We have shown that even at very low multiplicities of infection in recA- bacteria, the number of infective centers is not much lower than that for wild type (Table 2) and enough parental phage DNA is sufficiently intact to serve as template for significant amounts of new synthesis (Figs 2 and 3). Furthermore, we and others (Sironi et al., 1971) have been DNA in unable to detect significant breakdown of 32P-labeled parental red-gamthe recA- host. Moreover, the results shown in Figure 5 indicate that newly synthesized phage DNA is also quite stable. Thus, we conclude that large-scale, non-specific degradation of intracellular phage DNA does not contribute to the fee- phenotype. How, then, in the gam- infection can the BC nuclease cause the observed alteration in the replication program of /\? We propose (1) that the BC nuclease complex can interfere with the early to late transition through limited attack on critical (transient?) replication intermediates, and (2) that the gam protein circumvents this potential interference by inhibition or “control” of these nuclease activities, perhaps in a manner analagous to the recA product (Lecocq & Richelle, 1970). We presume from the known specificities of the BC nuclease (Wright et al., 1971; Goldmark & Linn, 1970) that the proposed intermediates possess either free ends, or extended single-stranded regions. Substrates not attacked by this enzyme in vitro include double-stranded covalently closed or relaxed circular molecules, the very forms which accumulate in gam- infection of BC nuclease+ cells. The action of the BC nuclease on these replication intermediates must be very limited and the damage quickly repaired, because in gam- infections DNA synthesis does not stop, but early replication continues throughout the lytic cycle. This fact suggests that the decision to replicate in an early or late mode may occur many times, perhaps at the end of each early round. It is possible that in a wild type infection, the number of early rounds depends on the concentration of gam product. An understanding of the special aspects associated with the termination of replication in circular molecules, namely the interaction of two converging replication forks (Kaiser, 1971), may provide clues to the mechanism of formation of the critical intermediates and to the factors which could influence the decision to replicate in the early or late mode. Comparison of gam- phages and the plasmid h dv (Matsubara t Kaiser, 1968) suggests parallels between their replication which may support our proposals. The circular X dv molecule presumably replicates solely in an early mode. This is reasonable in the present context because X dv is missing all of the genes in the early N operon, notably gam. It is also missing most late genes, but as Skalka (1971n) has shown, the late genes A through J are not required for late replication. The plasmids of X Nam7am53 (Signer, 1969; Lieb, 1970) and h dg in E. co&-Salmonella hybrids (Baron

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et al., 1970; Falkow & Baron, 1970) may be further examples of molecules locked in “early” replication due to gam deficiency. If the replication of gam- phages and the above mentioned plasmids is indeed analogous, we would predict that one might detect some late-type replication of plasmids in recA-Bhosts, provided of course that no essential genes (or sites) are missing or inactive in such plasmid chromosomes. Whereas it seems clear that one function of the gam protein involves inhibition of BC nuclease activity, the molecular basis of its other manifestations is not immediately obvious. The gam gene is apparently associated with recombination because its loss reduces red-mediated recombination threefold (Table 2 and Zissler et al., 1971). In addition, gam- (like red-) mutants fail to grow in polA- or other feb- hosts (Zissler et al., 1971). The involvement of gam in recombination, phage growth and control of BC nuclease suggests that X gam and the host recA protein may have similar and pleiotropic activities. (b) red and ret functions The role of recombination functions in both phage and bacterial development is not well understood. It is clear that in their absence growth is generally impaired (Signer, 1971). Our results (see Table 7) show that the rate of )I replication is abnormally low in the absence of the red function, in ret + as well as ret- (A- or A-B-) hosts (Pig. 4). This fact indicates that the h red proteins play a role in phage replication for which the host ret proteins cannot substitute. The red- defect in rate results in a decrease in the total amount of intracellular phage DNA synthesized (Fig. 3). Nevertheless, red- DNA preparations seem to contain a large proportion of normal structures (i.e. concatemers and linear monomers) and red- DNA is matured and packaged with good efficiency. Recall that in the recA- host, where the gam mutation results in an inhibition of late replication, absence of red proteins caused a defect in phage production (Table 2) despite the fact that late mRNA (and presumably late protein) synthesis was normal (Table 6). Our data (Fig. 6) indicate that the defect in phage production is correlated with an inability to produce DNA concatemers. Since such a defect is not obphage in the ret + host, we conclude that in this case red and served with red-gamret functions are equivalent. These facts lead us to propose (as has been independently suggested by Stahl et al. (1972)) that the concatemers contain a structure which is required by the phage morphogenesis system. Perhaps the DNA maturation enzyme(s) must see two cohesive-end sequences in the same molecule (Szpirer & Brachet, 1970). Normally, the required concatemers would be formed via late replication. However, in the absence of “late ” replication (i.e. in gain- infection of BC nuclease+ cells) they could be formed by ret or red-mediated recombination. Thus, the recombito conoatemer production that bypasses a nation functions provide a “shunt” potential block in DNA maturation. There are at ieaat three ways in which such functions could promote concatemer formation. The fust possibility is chown diagramatically in Figure 11 as a recombination shunt (a). We suppose that in this case the functions act to convert single monomeric circular molecules to multimeric molecules with no extended single-stranded regions and no free ends (requirements for co-existence with the BC nuclease). Because these multimeric circles contain at least two cohesive end sequences, they may be formally equivalent to concatemers produced by late replication. This interpretation is supported by the work of Stahl et al. (1972), who suggest that two or

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more unreplicated circular molecules can recombine and be matured wuiasuch a shunt, bypassing both early and late replication routes. Further evidence for circle to circle recombination comes from an apparently analogous situation. Hobom & Hogness (as quoted by Kellenberger-Gujer (1971)) suggested that such interactions occur frequently between h dv genomes and, more importantly, these events are ret dependent. If shunt pathway (a) is a major route, then most phages produced in the gaminfections should be red- or ret-mediated recombinants. (It is improbable that intmediated recombination plays any role because Ohere are no more phages produced on infection of recA- cells with int +red-yampoint mutants than with X bioll (bat-red-yam-) phage.) One might suppose, therefore, that the frequency of genetic mixing would be very high among progeny from a cross of two suitably marked yamphage. Our own results (see Table 1) and those of Zissler et al. (1971) show that this is not the case. In every host tested, the frequency of general recombination is slightly lower for yam- than for yam+ phages. If these differences are significant, we must assume that the frequency of genetic mixing does not reflect directly the frequency of molecular interactions (for example, there may be many “sister” interactions). Alternatively, another mechanism can be proposed (recombination shunt (b) in Fig. 11) that might generate concatemers from parental templa’tes through formation of new replication forks (Boon & Zinder, 1969). We imagine that the intermediates of shunt (b) must also contain structures that are not attacked by the hosts’ BC nuclease. The molecular interactions which may give rise to such forked structures have been summarized by Clark (1971). Finally, as noted in section (a) above (see Fig. 5(d)), our data suggest that red proteins can partially repair some of the damage incurred through the presumed action of the BC nuclease. Therefore, a third way in which recombination functions might promote concatemer formation is through actual repair of damaged replication intermediates. Some concatemers might arise, therefore, by partial “leakage ” of late replication. If, in the absence of normal replication, concatemer formation via recombination is essential for maturation, the few infectious phages that are produced in red-gamrecA - infections may reveal still another, perhaps less efficient, recombination system. Alternatively, and perhaps more likely, their production may signify that the concatemer requirement is not absolute. That is, occasionally mature DNA structures may be formed from molecules containing less than two cohesive end sequences. Such leakiness could also account for the occasional production of phages containing DNA with one mature end (Little & Gottesman, 1971; Yarmolinsky, 1971). As stated in the introduction, Skalka et al. (1972) could not exclude the possibility that concatemers were produced by some “hypothetical ” recombination system. We now consider the evidence relevant to the idea that the yam- mutation might define such a system. Genetic data (see Table 1 and Zissler et al., 1971), which show no significant general recombination among (yam+) red- phages in recA - or recA - Bcells, indicate that if the yam mutation does define an independent recombination system, it must be genetically invisible (e.g. specific for sites near the mature phage ends, like Ter (Yarmolinsky, 1971), or specific for “sister” molecules (Skalka, 1971a; Skalka et al., 1972)). Comparisonof the structure of DNA synthesizedunder conditions which eliminate the effects of both major general recombination systems (red- and red-game DNA from recA-Bcells, Table 7 and Fig. 9) indicates that the “hypothetical” recombination system must be physically invisible too. The yam- DNA has no distinguishable phenotype in recA-Bcells; the single mutant DNA looks

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like that of wild type and red-gam- DNA like red-. A final possibility which can be considered is that a gam-dellned system may not be independent. One might imagine that gam. together with red or ret genes might define a new, genetically invisible recombination system. It seems unlikely that gam works with ret this way because red - (gam + ) DNA has the same physical properties in ret + , recA - and recA -B- cells (compare Figs 8, 9 and 10). We are unable to exclude the possibility that gam can work in this way with red proteins because the physical consequences of such an interaction would be obscured by red-mediated general recombination. (c) Further

implications

It may be possible to exploit knowledge of the interaction between gam and host structure and location of other repheating forms of phage DNA. For example, Wolfson et al. (1972) have reported that bacteriophage T7 replicates as a linear molecule. Therefore, to exist in the presence of the BC nuclease, T7 may also code for a gam-like protein. The reduction of BC nuclease activity in T4-infected cells (Tanner & Oishi, 1971) suggests that this bacteriophage may produce such a protein. Because X gam mutants (in BC nuclease+ cells) replicate continuously as circular molecules, they may provide an important advantage for the study of structural intermediat,es and controls of early replication. Our preliminary studies of red-gainintracellular DNA revealed the existence of circular monomers that could not be repaired by T4 ligase or T4 DNA polymerase (Table 5). These molecules might be a by-product of limited action of the BG nuclease on replication intermediates, or they may be formed during a step in the normal pathway of replication (Young & Sinsheimer, 1967,1968). Our data indicate that the break in these molecules occurs on either strand. If the molecules are normal intermediates, this fact could signify that early or late origins of replication are not strand specific (see also Skalka et al., 1972). We have speculated that the interruption in. these circles is non-repairable because of some unusual structure. Such a structure might consist of a single or doublestranded tail, a 3’-phosphate-ended chain or (in view of the fact that RNA has been implicated recently in DNA replication; Wiokner et al., 1972) a ohain which starts or ends with ribonuoleotides. All of these possibilities are now being tested. DNA synthesis in recA--infected Finally, results from our studies of red-gamcells show that it is possible to block all of the major pathways for concatemer production by a combination of known mutations. If further study should prove that conoatemers are an obligatory intermediate in /I DNA maturation, this feesystem will be useful for analysis of the mechanism of phage morphogenesis. BC proteins in the analysis of the intracellular

These studies were initiated with the help of Dr J. Zissler, who provided us with many of the critical phage and bacterial strains. We are grateful for his continuous and enthusiastic interest and for the benefit derived from many discussions with him throughout the course of this work. We also thank Drs T. Takano, M. Oishi and E. Signer for other bacterial and phage strains used in these studies. Dr C. Harvey and Dr M. Gefter were most generous in supplying us with various enzyme preparations and advice for their use. Patricia Dawson rendered technical assistance with skill and much patience. Kenneth McMilin, Robert Malone (and the “rest of Stahl’s laboratory”) criticized an early version of the manuscript and helped us to think more clearly about various facets of the relations between replication and recombination. L. Patrick Gage criticized the flnal manuscript.

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