Uncoupling of late transcription from DNA replication in bacteriophage T4 development

J. Mol. Biol. (1970) 54, 103-119

Uncoupling of Late Transcription from DNA Replication in Bacteriophage T4 Development S.RrvA-f, A. CASCINOSAND E.P. GEIDUSCHEK# Department of Biophysics Uraiversity of Chicago Chicago, Ill. 60637, U.S.A. (Received 1 June 1970) The conditions for uncoupling T4 late transcription from concurrent replication have been explored. Replication-late tr8nscription coupling is retained when viral DNA is not glucosylated. Coupling is also retained in bact.eria that have been pre-infected with phage T3. The latter are known to induce the synthesis of an enzyme that hydrolyses S-adenosylmethionine, the methyl group donor in DNA methylation. However, in order to demonstrate this effect of ligation on replication-late transcription coupling it is necessary to prevent nucleolytic degradation of unligated DNA. This has been done by introducing a gene 46 mutation. Thus .&Ygene 43 (DNA polymerase)-ts or am gene 30 (DNA ligase)-am gene 46 mutants have been found to make late messenger, tail fiber protein and lysozyme in the absence of continuing DNA synthesis. The parental, unreplicated DNA of these phages can also serve as a template for late transcription and concomitantly undergoes single-strand scissions. A model of DNA “competence” for late transcription is proposed on the basis of these observations. (1) Competent DNA contains interruptions (breaks or gaps) that are essential for the binding-initiation steps of T4 late RNA transcription. (2) Gaps are created by endonuclease action or as the direct consequence of discontinuous replication. (3) DNA ligase seals these interruptions and controls late transcription negatively.

1. Introduction The preceding paper (Riva, Cascino & Geiduschek, 1970) concerned itself with the extent to which late transcription is coupled to DNA replication during phage T4 development. It was shown that all enzymes necessary to late transcription can be synthesized in the absence of DNA replication thus suggesting that the coupling is probably due to the existence of an unstable “competent” form of DNA whose continuous supply is ensured by continuous DNA synthesis. The purpose of this work is to study the properties that determine competence for late transcription. We have approached this problem by trying to f%rd conditions for uncoupling replication from transcription in vivo. (This might also be helpful in the biochemical analysis of late messenger synthesis.) The experiments of the preceding paper can be explained by assuming that the unmodified freshly synthesized DNA is “competent,” and that some enzymic process controls late transcription negatively by modifying competent DNA. Accordingly, in this work, we have investigated the effects of several enzymic modifications that the viral DNA is known to undergo after 7 Present address: Miorobiology Department, Lepetit S.p.A., Vis Dumndo 38, Milano, It&y. $ Present address: Department of Biology, University of California, San Diego, L8 Jolla, Calif. 92037, U.S.A. 103

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replication (maturation (packaging of DNA), gluoosylation, methyl&ion, action of nuolease(s), ligation by DNA joining enzyme), on the coupling of late transcription to replication. The pattern of the experiments is similar to that of the preceding paper. DNA synthesis has been arrested by shifting a ts mutant in DNA polymerase to the non-permissive temperature. The chemical modifications mentioned above have been controlled either by ohlorampheniool (in the case of maturation) or by inserting the proper mutations. Multiple mutants have been constructed in which late transcription and DNA synthesis are uncoupled. With some of the latter mutants, parental DNA can be made competent to serve as a template for late transcription.

2. Materials and Methods Most of the materials and experimental techniques are described in the preceding paper (Riva et al., 1970). Unless otherwise noted, bacteria were infected with 8 to 10 phages/oell. (a) Phuge stocks The T4 mutants Table 1.

that

have not been described

previously

(Riva

et al., 1970) are listed

in

TABLE 1

Xingle and multiple T4 mutants

amNl30 MAdO

46 30

tap36 tap36 tap36 tsP36

43,30 43,30 43,e 43, wt, Bgt

amH39gt+ (H39X) tsA80 amM41 ama~gt (8)

tsP36 amH39X

amNl30

tsP36 @A80 amN130

J’ At DA

DNA polymerese, DNA polymerase, DNA polyrnerase, transferase DNA polymerase, exonuolease DNA polymarase, exonuclease

43,30,46 43,30,46

shgt, DNA

Exonuclease ? DNA ligase

DNA polymerase,

t DA, DNA (b) Temperature

Functions(s) missing under non-permissive conditions

Phenotype

Mutants

synthesis,

DNA ligase DNA ligase lysozyme u-and ,%glucosyl DNA ligase, DNA ligase,

arrested. and RNA-RNA

hybrid

analysis

Small volumes of culture (less than 30 ml.) were shifted to the non-permissive temperature as described in the preceding paper. When large volumes had to be shifted to the higher temperature (40°C), the culture at 30°C was quickly poured into an equal volume of was then medium vigorously aerated and pre-warmed at 60°C. The final solution quickly transferred to a 40°C bath. The temperature was continuously controlled during this manipulation. DNA synthesis was determined by measuring [14C]thymidine incorporation into 5% ClsCCOOH-precipitable material after overnight digestion in 0.5 M-NaOH. The varying speoifio activities of thymidine are specified in the text. RNA was labeled during 2 min with [3H]uridine (3 to 7 &ml.; 0.5 to 15 pgglml.). RNA-RNA hybrid formation and analysis was as described by Geiduschek & Grau (1970). (c) Cell 1ysL and DNA

preparation

for

C.&l artd sucrose density

These procedures more or less follow established methods to be described in detail elsewhere (Cascino et al., manuscript

gradient

centrifugution

(Frankel, 1966,196s) in preparation).

and are

T4

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105

3. Results (a) The search for means of uncoupling late transcription from replication In the preceding paper (Riva et al., 1970) the coupling of T4 late transcription to DNA replication was postulated to be due to an unstable, competent form of DNA whose modification-maturation controls late transcription negatively. The first question suggested by this model is the following: does late transcription-replication coupling require continuous protein synthesis 1 Suppose for example, that maturation of phage is the process which modifies the competent template. Since maturation (i.e. packaging of DNA into phage heads) is a stoichiometric process, continuous protein synthesis might be required for replication coupling of late transcription. In order to test this hypothesis, the following experiment was performed : E. coli BE were infected with tsP36 (gene 43) phage also carrying a lysozyme mutation; chloramphenicol was added 14 minutes after infection and the temperature was subsequently shifted up (15 and 20 min after infection). The result of the experiment is shown in Table 2: TABLE 2 Chloramphenicol does not uncouple late RNA synthesis jrona replication Time of labeling (min) la-20 25-27 33-35 21-23 23-30

Temperature labeling (“C) 30 42 42 42 42

of

Time of shift-up (min)

Percentage of RNA competed in DNARNA hybridization by 2 mg/ml. T4 wild type 20-min RNA but not B-mm RNA

20 20 15 16

42 9.5 3.7 8.5 4.5

E. coli BE were infected with T4 tsP36 (gene 43) amM41 (lysozyme). Chloremphenicol (100 pg/ml.) w&s ctdded 14 min after infection. Cultures were shifted to 42”C, 15 or 20 min after infection and labeled for 2 min at various times before and after shift-up.

continuous protein synthesis is not required in order to ensure the coupling between replication and late transcription. If an enzymic process is responsible for the modification of DNA against late transcription it is likely to be an early function. Among the modifications that T4 DNA is known to undergo upon replication, ligation, glucosylation and methylation are induced by early enzymes. The following experiments were designed to determine whether the replication-late transcription coupling does require glucosylation or methylation. Glucosylation or methylation of DNA were controlled by infecting with appropriate mutants and then performing the standard temperature-shift experiment. The effect of preventing glucosylation of newly replicated DNA was tested by using the mutant tsP36 arnufigt(8). This mutant grows normally at 30°C in E. coli K12, A564 (su-, T-raST4,r&J but late messenger synthesis shuts off very quickly after shift to the non-permissive temperature (see Fig. 9 of the previous paper). The connection between DNA degradation and complete late messenger shut-off upon shift-up has been noted in the previous paper (Riva et al., 1970). The experiment in which methylation was manipulated is described in Figure 1. It has been shown that preinfection with ultraviolet-inactivated T3 phage prevents T4 DNA methylation by

106

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Time (min)

Fm. 1. DNA and late messenger synthesis in E. coli BE infected with UP36 following preinfection with ultraviolet-inactivated T3 phage. T3 phage were kindly provided by P. Siersma and were inactivated with ultraviolet light to about 10-s survivors. 10 min before T4 infection, cells were infected with 6 T3 phages per cell; at t = 0 min cells were superinfected with tsP36 and the temperature shift was performed at the time shown. DNA synthesis and late messenger analysis were performed as previously described (Riva et al., 1970, Fig. 1). -a-@-, Thymidine incorporation at 30°C; -- 0 -- 0 --, thymidine incorporation at 42°C; the arrow indicates the time of shift-up; histogram: blank area, RNA labeled at 30°C just before shift-up; shaded area, RNA labeled at various times after shift to 42°C.

inducing the synthesis of a T3 viral enzyme that splits S-adenosylmethionine (Gefter, Hausmann, Gold & Hurwitz, 1966). Cells were preinfected with five ultraviolet killed T3 phages per bacterium and ten minutes later infected with tsP36. In this case late messenger synthesis is also decreased after shift to the non-permissive temperature. The result argues against an essential role of methylation in controlling the competence of DNA for late transcription. There is considerable evidence that DNA is extensively cleaved by endonucleases and repaired by ligase during replication (Kozinski, 1968). Furthermore, the Okazaki model of replication postulates a discontinuous synthesis of DNA with the intervention of other enzymes, among them the phage-coded DNA ligase, to produce complete DNA molecules (Okazaki, Okazaki, Sakabe & Sugimoto, 1967; Okazaki, Okazaki, Sakabe, Sugimoto & Sugino, 1968). Some of these steps of replication could generate competent template for late transcription while others could modify DNA against competence. The possibility that DNA ligation might directly or indirectly affect the ability of DNA to sustain late transcription is suggested by the following observations : (1) T4 ligase amber mutants (gene 30) accumulate very little progeny DNA (Hosoda, 1967; Bolle, Epstein, Salser & Geiduschek, 1968) but are able to sustain an almost normal proportion of late messenger synthesis (Bolle et al., 1968). It has been shown that these mutants continue to synthesize a labile form of T4 DNA and that they produce about 20% of the quantity of late proteins made by T4 wild type (Hosoda, 1967; Hosoda & Levinthal, 1968). (2) Temperature-sensitive ligase mutants, when shifted to the non-permissive temperature, drastically reduce the rate of net incorporation of [14C]thymidine into DNA. Nevertheless, shifting such mutants to 43°C does not quickly produce differential effects on late transcription (Fig. 2). The following hypothesis could therefore be made: late transcription occurs only on a

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107

50 40 ; 30 a

20 E 0

IO 4 b 0 Time (min)

FIQ. 2. DNA and late messenger synthesis tsA89 (gene 36)-infeoted cells. Experimental pper. Symbols as in Fig. 1.

in a temperature-shift experiment performed with conditions as desoribed in Fig. 1 of the preceding

competent template, which has single strand breaks. These breaks are constantly being sealed by phage ligase and are created by new replication (or they are created by endonuclease action in newly synthesized DNA only). Under normal phage development conditions, a steady pool of competent DNA is created by this balance of synthesis and maturation. After the arrest of DNA synthesis, however, this pool is depleted as ligase action continues. In order to test this hypothesis, we attempted to fix the state of DNA competence by making DNA template inaccessible to ligase action after shift-up. The tirst two experiments were carried out with the following two mutants: (1) tsP36 amH39X (genes 43, 30) ; with this mutant we hoped to accumulete competent template at 30°C and to retain it after shift-up. (2) tsP36 tsA80 (genes 43, 30) ; in this latter mutant we hoped to have normal DNA synthesis

Time (min)

FIG. 3. DNA and late messenger synthesis in tsP36 antH39X-infected

cells. The procedure for measuring oontinuous [Wlthymidine incorporation, for labeling the RNA and for meaaring the percentage of late messenger are the same as those described in Fig. 1 of the prece&va9 mr. [W]Thymidine (O-06 PC/ml.) and 20 pg cold thymidine/ml. were added 3 min after infection. No cell lysis was observed during the experiment. Symbols as in Fig. 1.

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at 30°C and then “freeze” this DNA by sufficiently rapid inactivation of both DNA pol ymerase and ligase ; rather than accumulating competent template, we would preserve only the fraction of competent template that was available for synthesizing late messenger at the moment of shift-up. The results of the experiments with these mutants are entirely contrary to these expectations (Figs 3 and 4). In both cases late messenger synthesis drops very quickly I

I

I

I

Time (mid

FICA 4. Temperature shift (30°C to 42’C) after infection

with the double mutant taP36 tsAd0 (genes 43,30). Details as in Fig. 3 and Fig. 1 of the preceding paper. The significance of the instability of DNA after shift-up is discussed in the text. Although some authors (Newman & Hanawalt, 1968) have only observed a complete inactivation of tsA80 DNA ligase at a higher temperature than 42°C (43 to 44”C), we obtained similar degradation at 42°C and at 43*5”C.

following phage DNA synthesis arrest. We tentatively attribute the failure of these experiments to the instability of the template synthesized in these conditions. The degradation of the template in the case of tsP36 amH39X mutant could be due to the instability of the low molecular weight DNA made by this mutant (possibly the SOcalled Okazaki pieces). The result of the experiment with the mutant tsP36 tsA80 however came as a surprise; it appears that DNA synthesized under permissive conditions becomes unstable upon inactivation of ligase and of DNA polymerase. Another ts polymerase-ligase mutant tsP36 tsB20 (genes 43, 30) has properties that are identical with those of tsP36 tsA80 (data not shown). Ligase action seems therefore to be continuously required for the stability of phage DNA and this implies that viral DNA is subject to continuous action of nucleases (cf. Kozinski, 1968; Hosoda & Mathews 1968,197O). It has been shown (Hosoda t Levinthal, 1968) that degradation of DNA in ligase mutants to acid-soluble oligonucleotides can be prevented by introducing a second mutation in gene 46 (or 47, or both). Genes 46 and 47 determine an exonucleolytic activity that is involved in host DNA degradation (Wiberg, 1966) and in phage recombination (Bernstein, 1968). The phenotype of gene 46;or 47 am mutants is DNA-

T4

REPLICATION-TRANSCRIPTION

UNCOUPLING

109

arrested (DA) and the phage yield of these mutants in the su- host is not negligible (about 10 phages/cell) while the proportion of T4 late messenger synthesis is some what less than normal (Bolle et al., 1968; Guha et al., manuscript in preparation). We therefore constructed the two mutants tsP36 amH39X amN130 and tsP36 tsA80 amNl30 (genes 43, 30, 46) and performed the standard shift-up experiment on cells infected with these triple mutants. DNA synthesis in the shift-up experiment clearly shows the effect of the gene 46 mutation; little or no degradation of DNA to acid-soluble nucleotides is observed after shift-up, and the pattern of late messenger synthesis after shift-up is changed (Fig. 5).

Time (min)

FIG. 5. Temperature shift after infection with triple mutants in genes 43, 30 and 46. (a) tsP36 amH39X amN130 : [14C]Thymidine was added at 3 min after infection (0.0’7 &ml. ; 15 pg/ml.); RNA was pulse-labeled with [3H]uridine for 2 min (5 PC/ml.; 1 pg/ml.). Shift was from 30 to 40.5’C. Symbols as in Fig. 1. (b) tsP36 hAdO amNl30: Shift was from 30 to 42°C; labeling conditions as in (a).

Late transcription is not shut off, as in all previous experiments ; instead, the proportion of late RNA remains about the same for more than ten minutes after the shift-up. In a control experiment with double mutants tsP36 amNl30 (ts gene 43, am gene 46) late messenger synthesis is shut off after shift to the non-permissive temperature (data not shown). Thus the existence of all three mutations is essential to demonstrating this uncoupling of late transcription from replication. Although the proportion of late messenger synthesis in cells infected with these mutants is rather low compared to the wild type (compare Fig. 5 with Fig. 1, preceding paper) the late messenger that is made, appears to be physiologically active and this is shown by measuring production of T4 lysozyme and of the gene 34 product. As Figure 6 shows, the synthesis of these two late proteins is enhanced by the shift to 40°C in cells infected with T4 tsP36 amH39X amN130 or by the shift to 42°C in cells infected with tsP36 tsA80 amNl30. The absolute amount of protein synthesized is, however, about five times lower than in wild-type phage-infected cells (data not shown) and the rates of synthesis of these proteins decline 30 minutes after infection. These seem to be general properties of ligase-deficient mutants (Hosoda & Levinthal, 1968 ; Riva, unpublished observations) ; we attribute tentatively the non-differential decline of ability to make viral proteins to the fact that the DNA in these mutants appears to be seriously damaged (Hosoda & Mathews, 1968,197O) and that its ability

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I 20

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I

Time (mid

FIG. 6. Late protein synthesis after temperature shift with E. co& infected with a T4 gene 43, 39, 46 triple mutant. (a) tip36 amH39X amNl30: Lysozyme synthesis at 30°C and after shift to 4O’C; the extracts were prepared as described in the preceding paper; the lysozyme concentration is expressed in arbitrary units per 2 x lo7 cells (this arbitrary unit has been defined in the preceding paper). (b) taP36 tsA80 amNl30: Lysozyme synthesis at 30% and at 42°C; experimental conditions as in (a); the temperature was shifted to 42°C rather than 40°C in order to inactivate the tsA30 gene 30 product as well as the tsP36 gene 43 product. (c) Gene 34 product synthesis at 30°C and at 40°C after infection with tsP36 amH39X amNl30: the concentration of gene 34 product is expressed in phage equivalents/ml.

to sustain messenger synthesis is generally reducedt. It is interesting to note that both am and ts ligase mutations give the same effect in uncoupling late transcription from replication. This adds generality to the observation: in order to retain the template competent for late transcription, ligase function may be absent either during the entire infection or starting from a certain time (of shift-up to a non-permissive temperature). We have shown (Casoino, Riva & Geiduschek, manuscript in preparation) that viral DNA in tsP36 amH39X arnNl3Pinfected cells is subject to some degradation at 3O"C,although semiconservative replication still takes place on this template. This raises an interesting question about the physical properties of the RNA synthesized on such templates. It is possible that the unligated DNA templates might lead to abnormalities of transcription, even though a part of the late messenger is obviously able to direct synthesis of functional proteins. Do nicks in the template lead to abnormal RNA chain initiation in vivo 1 If this were so, then synthesis of nonsense t We do not know whether the inactivation is uniform for all viral proteins. It might be differentially effective for smaller and larger proteins and for the products of genes with different locations relative to their oonjugate promoters.

T4

REPLICATION-TRANSCRIPTION

111

UNCOUPLING

anti-messenger might occur in v&o. In fact, it has recently been shown that even wildtype T4.infected cells synthesize mutually complementary RNA species at different times of the viral development (Geiduschek & Grau, 1970). The hypothesis has been put forward that this situation arises because of a virus-induced change in the mechanism of RNA chain termination during T4 infection (Brody & Geiduschek, 1970). RNA labeled at various times after infection by various mutants and T4 wild type has been amesled with T4 wild type unlabeled 6. and 20.minute (early and late) RNA and tested for the formation of RNA-RNA duplexes resistant to pancreatic and T, RNases (Table 3). The main result of this analysis is that RNA synthesized in TABLE 3 RNA-RNA

Labeled

duplex @ma.tion Percentage RN&se resistance with 0 or 500 M unlabeled wild-type RNA/ml.

RNA Time of shift to 40 or 42°C

Phage

Time of labeling (dn)

Temp. of labeling (“C)

None

5.min RNA

IO-min RNA 1.4 5.0

Wild type Wild type Wild type

None None None

l-5 17-20 48-53

30 30 30

0.3 3.5 1.6

o-4 23.9 32-O

6.1

tap36 amM41

None 22 min

20-22 33-35

30 40

1.2 5.3

26 11-7

l-2 67

amH39X

None

17-20

30

5.4

15-2

5.3

None 20 min None 17 min None

la-20 28-30 15-17 23-25 28-30

30 40 30 40 40

1.5 3.3 1.7 2.7 <1*

10-5 18.4 9.7 15.6 6.5*

3-3 10.4 3-3 7.5 3.5*

tsP36 amH39X

amNl30

Annealing was at 70°C for 3 hr in 0.4 M-N&I, 0.08 M-Tris Cl (pH 7*5), 0.004 M-EDTA except for samples marked with an asterisk which were annealed at 60°C for 6 hr. Under the latter conditions, the efficiency of RNA-RNA duplex formation is reduced by approximately 40%. Subsequent digestion was at 37°C for 60 or 120 min in 0.12 aa-NaC1, 0.023 r-r-Tris Cl (pH 7.5), O-01 M-MgCl,, 0.0008 M-EDTA with 10 fig pancreatic RN&se/ml., 10 pg pancreatic DNese/ml. and 1 pg T1 RNase/ml. The concentration of labeled RNA varied from 7 to 40 pg/ml. except for the amH39X RNA which w&s tested at 200 pg/ml.

ligase mutant-infected cells (including the gene 46,43,30 triple mutants) is neither remarkably symmetric nor remarkably different in anti-messenger content from corresponding wild-type RNA. There are minor differences between the mutant RNA’s however, and these are understandable in terms of the following properties of wildtype RNA: (a) The anti-20.minute RNA content of T4 wild type RNA is low (1.5 to 7%) at all times of infection. A substantial increase above this level is probably due to anti-late messenger synthesis. (b) Five-minute wild-type RNA converts a large fraction of wild-type late RNA to RNase resistance (Table 3, lines 2 and 3, column 6). The fraction of labeled wild-type late RNA converted to RNase resistance by 500

112

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pg B-minute RNA/ml. ia 40 to 50% of r-strand transcription (cf. Geiduschek BEGrau, 1970 ; Guha, Salser & Szybalski, 1968). Some labeled mutant RNA, tsP36 amH39X amN130 RNA made after shift to 40°C contains slightly more anti-ZO-minute RNA than the wild type (compare Table 3, lines 8 and 10) and the fraction of anti- 20-minute RNA increases after shift to 40°C. However, even if this is anti-sense RNA, it is only a small fraction of all the T4 RNA made after shift to the non-permissive temperature. The anti-5-minute content of this RNA also increases after shift to the non-permissive temperature, while the fraction of late transcription remains approximately the same (Fig. 4) and this probably reflects the presence of another small fraction of anti-messenger. The RNA made in tsP36 amH39X amNl30-infected E. coli BE continuously at the nonpermissive temperature evidently is low in all kinds of anti-messenger. The RNA made in T4 ligase am mutant-infected cells (Table 3, line 6) is not very different from the corresponding wild-type late RNA. It is possible that differences in the relative abundances of late RNA species could exist and that some might not be synthesized at the non-permissive temperature after shift-up of tsP36 amH39X amNl30-infected cells. However, a hybridization competition experiment shows that these cells contain all late messenger species that are present in wild-type infected cells in the absence of viral replication, although presumably at lower concentrations. Cells (500 ml.) were infected with the triple mutant at 30°C; 17 minutes after infection, the temperature was quickly raised to 40°C and after 10 minutes at 40°C (t = 27 min), cells were harvested and RNA extracted. This RNA was then used as a competitor in the mixed-competitor experiment shown in Figure 7. It can be seen that RNA of the triple mutant is able to compete T4 wildtype late RNA very completely and so contains all RNA species present in wild-type

col

Unlabeled

RNA(mg

/ml )

(b)

Pm. 7. Mixed competitor test for late messenger content of RNA extracted from E. co& infected with mutants tsP36 amH39X amNl30 (genes 43, 30, 46) and tsP36 amM41 (genes 43, e). (E) Hybridization: all samples contained 10 pg denatured T4 DNA/ml., 4 pg T4 am+ RNA/ml. labeled from 17 to 20 min after infection and: curve A, increasing amounts of 5-min unlabeled wild-type RNA competitor; curve B, a constant amount of unlabeled 5-min wild-type RNA (1 mg/ml.) and increasing amounts of RNA extracted from taP36 amH39X amNl30-infected cells 27 rnin after infection (the culture was shifted to 40°C at 17 min, see text); point C (0) 1 mg/ml. of unlabeled 6.min and 1.2 mg/ml. of unlabeled wild-type 20-min RNA. (b) Hybridization conditions as in (a). Curve A, increasing amounts of unlabeled 5-min wildtype RNA; curve B, 1 mg/ml. of ELmin unlabeled RNA and increasing amounts of tap36 amH39X amN130 RNA (extracted 30 min after infection at 40%); curve D, 1 mg/ml. of unlabeled 5min RNA and increasing amounts of RNA extracted from tsP36 amM41 30 min after infection (the culture was shifted to 40°C at 20 min after infection) ; point C (0) same as in (a).

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late RNA. For a comparison, the cold RNA extracted after the temperature shift with the mutant tsP36 amM41 (genes 43, e) was also used. The competing strength of this latter RNA is much lower, although not negligible (Fig. 7(b)). A pert of the competing power of both RNA’s could be due to RNA that has been made before the shift-up and that is not entirely degraded. We have pointed out that a small fraction of normal late messenger synthesis is replication uncoupled (see Rive et al., 1970). (b) Parental DNA as template for late transcription When E. coli are infected with tsP36 arnH39X amNl30 at 4O”C, phage DNA is not made (Cascino et al., manuscript in preparation) but late messenger appears about 15 minutes after infection, reaches a maximum at about 30 minutes after infection and then decreases (Fig. 8). This RNA (labeled 28 to 30 min after infection) is not symmetric (Table 3). Lysozyme and gene 34 protein are synthesized with the kinetics shown in Figure 8. Comparable results have been obtained with the mutant tsP36

I

I’

,’ I’ I’

40-

/-

.’

I’

b c= 30:: E 20Y 4 IO8 0

IO

20

30

n ’10

-( 50

Time (min) FIQ.

8. Late messenger and late protein synthesis after infection

of E. coli BE with taP36 arnH39X

amN130tlt40°C. (a) Cells were grown to 5 x 1 On/ml. in M9S at 30°C. 10 min before infection the temperature w&s raised to 40°C md at, t = 0 min, oells were infeoted with 6 phages per cell and infeation oarried out et 40°C. After 1 min there were leaa than 1 y0 surviving cells. Late messenger, lysozyme ctnd gene 34 Lysozyme (arbitrary units); --@--a--, produot were assayed as already described. -O-O-, gene 34 product.

tsA80 amNl30 (data not shown). It appears therefore that in these mutants, parental DNA becomes the template for late transcription by a mechanism that does not require DNA replica;tion. The switch from early to late functions, however, seems to take place very late (compared with wild type at 40°C) as though the absence of T4 DNA synthesis slowed down a process that in normal conditions is accelerated and amplified by replication. When parental DNA serves as template for late transcription the synthesis of late messenger and of late proteins is dependent on the multiplicity of infection, as the following experiment shows. Bacteria were infected at 40°C with different multiplicities of purified tsP36 amH39X amN130, tsP36 and wild-type phage. Lysozyme and gene 34 product were assayed 40 and 50 minutes later (Table 4). Two minutes after infection, 200 pg chloramphenicol/ml. was added to a portion of each infected culture, 8

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AND

E. P. GEIDUSCHEK

and the levels of these two proteins in the continued presence of ohloramphenicol were taken as a measure of background. The amount of these two late proteins was found to increase with the multiplicity of tsP36 amH39X amNl30 infection but less than 1O-3 viable progeny phage per cell were produced after 60 minutes at 40°C. The known absence of multiplicity dependence of lysozyme and gene 34 product synthesis in T4 wild-type infection was confirmed in a control experiment. In another control experiment little or no lysozyme or gene 34 product synthesis was found when cells were infected with tsP36 (gene 43) at 40°C and low multiplicity, but at the highest multiplicities, lysozyme and gene 34 product synthesis were clearly detectable (Table 4). A low but detectable rate of lysozyme synthesis has been observed late after E. coli B infection with various T4 am replication-defective, DO, mutants (Mark, 1965; Kutter & Wiberg, 1968). It is conceivable that this weak and markedly multiplicity-dependent expression occurs on DNA templates that achieve a low level of competence as a result of recombination?. The competing power of the messenger RNA extracted 30 minutes after infection at 40°C with 8 phages/cell was measured as described above. The results are shown in Figure 7(b). It can be seen that the competing power of this RNA is somewhat lower than that of the RNA extracted after shift-up of the same mutant from 30 to 40°C but very much higher than that of the RNA extracted after shift-up with tsP36 amM41. The fate of the parental and progeny DNA and their physical properties after infection at 40°C and at 30°C were studied. 16N,13C density- and 3H radioactively labeled tsP36 amH39X amN130 phage were used to infect bacteria in light medium at 30 and 40°C and labeled with [14C]thymidine. The DNA extracted from these cells was examined by isopycnic and velocity centrifugation. (The detailed experimental methods and results will be described elsewhere; Cascino et al., manuscript in preparation.) Replication of parental DNA, detected by density shift of parental label, occurred at 30°C but not at 40°C. Neither was repair-incorporation of [14C]thymidine into parental phage DNA without density shift detected at 40°C (although incorporation into host DNA was observed; Cascino et al., manuscript in preparation). The size of the parental DNA re-extracted from infected cells was also examined by neutral and alkaline sucrose gradient centrifugation. There are marked differences in the fate of parental DNA after infection at 30 and 40°C. At 3O”C, helical parental and progeny DNA have a lower sedimentation coefficient than mature T4 DNA; at 40°C most of the parental DNA sediments with the mature T4 DNA marker and therefore does not contain double-strand breaks. Evidently, the double-strand breakage of parental DNA is concomitant with replication. There are simple ways in which this could occur and the question will be considered in more detail elsewhere (Cascino et al., manuscript in preparation). Upon alkaline denaturation, the parental DNA from the cells infeoted at 40°C has a wide distribution of sizes, but an appreciable fraction of it is in small fragments (Fig. 9, open circles). Thus the unreplicated parental DNA, though not subject to double-strand breakage, contains many interruptions in the individual DNA strands. In experiments, the details of which will be published elsewhere, we have shown t Alternatively, it may be that the late protein synthesis in t8P36 infection reflects the same process that occurs in the tsP36 amH39X amNl30 infection. Normally, mutations in gene 30 depress late fimctions by a factor of five (Hosoda & Levinthal, 1008) and gene 46 mutations also depress late functions. In the absence of replication, the opposite is the case.

amN130

(0.09) 17.5 26.0 (0.04) o-14 0.24 (0.08) 0.9 1.0

40 min 40 min 50 min 40 mm 40 min 50min 40 min 40 min 50 min

+ Cam

+ Cam -Cam

+ Cam

-Cam

3.3

of infection

Multiplicity

- Cam

4

(0.35) 2.7 3.0

(0.18) 0.32 0.54

(0.09) 17.5 27-o

Lysozyme 11.7

(0.20) 3.0 4.0

(0.18) l-28 1.84

(0.10) 20.0 31.0

23.4

(O-08) o-4 0.3

(0.08) o-1 0.1

IO-9

‘;‘y’

3.3

(0.29) 1.0 1.8

(0.52) o-1 0.3

(O-29) 4.2 8.3

Gene 34 product 11-7

(0.37) 2.0 3.2

(O-63) 0.8 1.8

(0.66) 5-l 10.3

23.4

Lysozyme and serum blocking power are expressed in the same units as in Fig. 6. All infections were at 4O“C. Chloramphenicol, where present, was at 000 pg/ml. As stated in the text, the + Cam values (in brackets) are regarded as background and have been subtracted from the -Cam values shown. Abbreviation used: Cam, chloramphenicol.

tsP36 amH39X

hP36

T4 wild type

TABLE

Lysozyme and gene 34 product synthesis without replication

116

S. RIVA,

A.

CASCINO

AND

E.

Fraction

number

I’.

GEIDUSCHEK

FIQ. 9. Single-strand breaks introduced into infecting T4 DNA in the absence of replication. (-O--O--) E. co& BE were infected with 8 3H-labeled phage tsP36 amH39X amN130 per bacterium at 40°C. 15 min later, cells were lysed (Cascino et al., manuscript in preparation); the lysate was layered onto a 5 to 20% sucrose gradient in 1 M-NaCl, 0.2 M-KOH and centrifuged at 15°C for 150 min at 1.3 X lo5 g,,, (Beckman SW39L rotor, 35,000 rev./min). Fractions were collected from the bottom of the tube and precipitated in the presence of oarrier denatured salmon DNA with 5% ClsCCOOH, filtered and counted. Sedimentation is from right to left. (-X-X-) Alkaline sucrose gradient oentrifugation of DNA from the purified phage.

(Cascino, Riva & Geiduschek, 1970) that late gene expression on either newly replicated or parental DNA is under the control of T4 genes 33 and 55, the so-called “maturation” genes (Epstein et al., 1963; Bolle et al., 1968; Pulitzer, 1970).

4. Discussion In the preceding paper we have shown that late transcription is continuously, and almost completely, coupled to phege DNA replication. The search for conditions under which these two processes are uncoupled was made under the assumption that although other hypotheses presented themselves, some kind of chemical modification would ultimately be found to control the ability of newly replicated DNA to work as template for late messenger synthesis and that the search for uncoupled late transcription would provide some insights into the mechanism of replication-late transcription coupling. The results of the first experiments reported in this paper rule out the possibility that glucosylation or encapsulation might be directly involved in controlling late transcription and indicate that methylation also is not involved (Fig. 1 and Table 2; also Fig. 9 of the preceding paper). We have, however, found conditions for uncoupling late transcription from DNA replication by eliminating the functions of two genes: gene 30 (phage ligase) and gene 46 (presumptive phage-coded exonuclease) ; with phage mutants that meet these specifications, late messenger and late protein synthesis do not decrease after DNA synthesis arrest (Figs 5 and 6). Furthermore, late transcription can take place, even in the absence of any DNA replication, on the parental DNA (Fig. 8). The messenger synthesized under these conditions appears to be physiologically active and we have been unable to detect any major anomaly in its physical characteristics or in the relative abundance of different transcripts. Therefore, although we cannot exclude the possibility that other mechanisms for uncoupling late transcription from replication exist, we proceed on the assumption that this mechanism is likely to have some biological relevance. What are the mechanism(s) that involve the gene 30 and 46 products with late transcription? Consider, first, what is thought to be known about the role of these two gene products in DNA replication.

T4

REPLICATION-TRANSCRIPTION

UNCOUPLING

117

Gene 30 codes for the T4 DNA ligase. Okazaki et al. (1967, 1968) proposed a discontinuous mechanism of in vivo DNA replication in which short DNA segments, the “Okazaki fragments” (sedimentation coefficient approximately 8 s) serve as intermediates in the formation of longer DNA chains. The role of DNA ligase in this model is that of joining short, primary DNA segments (Okazaki et al., 1968; Hosoda & Mathews, 1968; Newman Q Hanawalt, 1968; Richardson et al., 1968). That the T4 gene DNA ligase does not have a qualitatively essential role in T4 replication is suggested by the observations that r.lI mutations are specific suppressors of ligase mutations (Karam, 1969; Kozinski Q Mitchell, 1969; Chan, Shugar & Ebisuzaki, 1970) and that progeny DNA and viable phage can be produced in ligase amber mutant infection after interrupting viral replication during a critical period. The mature T4 DNA synthesized under such conditions does not have the normal stability (Cascino et al., manuscript in preparation). A different primary role for the gene 30 T4 ligase has been suggested by Kozinski (1968) : the repair of single-strand breaks produced by a phage-coded endonuclease. In fact, recent experiments (Hosoda & Mathews, 1970) indicate that two distinct classes of DNA fragments exist during gene 30 ligase-less T4 replication-8 s and 12 to 16 s pieces. The 8 s pieces are the primary products of DNA polymerization and are precursors of longer DNA chains with or without T4 ligase (Kozinski, 1968). The 12 to 16 s pieces can, of course, be rejoined into longer chains but are not direct products of in vivo DNA polymerization. There is some, though not conclusive, evidenoe that they may be created by endonucleolytic cleavage from larger DNA chains. The genes 46 and 47 control nuclease functions that are involved in host DNA degradation and in phage DNA recombination (Wiberg, 1966 ; Bernstein, 1968; Kutter & Wiberg, 1968), but there is no evidence yet that the product of either gene is part of a nuclease (Wiberg, personal communication). Some relevant properties of mutants in genes 46 and 47 are: (a) Mutations in gene 46 are known to prevent host DNA degradation without preventing its fragmentation (Wiberg, 1966; Kutter & Wiberg, 1968). (b) The gene 46-, 47-associated nucleolytic activity also acts on T4 DNA, since mutations in gene 46 prevent the solubilization of the T4 DNA synthesized by amH39X (gene 30)infected cells (Hosoda & Levinthal, 1968). (c) However, am mutations in gene 46 do not prevent the accumulation of single-strand breaks in parental or newly synthesized DNA in T4 ligase-less infection. (d) A mutant in gene 46 is unable to form concatenated DNA molecules (Hosoda & Mathews, 1970). On the basis of the above considerations, a model of late transcription-replication coupling can be proposed. In this model the T4 DNA ligase has an important role. We propose that DNA becomes “competent” for late transcription through the action of an endonuclease immediately following replication or as the result of replication itself. Competence presumably constitutes the creation of sites for binding RNA polymerase and/or for the initiation of late RNA chains. In the preceding paper, the hypothesis, that the transcribing enzyme which is active at these sites is not active for any other viral transcription, was put forward. The interrupted structure of DNA is an essential element of competence. The phage-coded DNA ligase seals the breaks, making the template incompetent for late transcription. DNA replication continuously supplies new template which contains interruptions and also undergoes extensive endonucleolytic cleavage, and which is subject to repair by DNA ligase. When DNA synthesis is arrested and ligase action is allowed to continue (for example,

118

S. RIVA,

A.

CASCINO

AND

E.

I?. GEIDUSCHEK

by shifting cells infected with tsP36 to the non-permissive temperature) all the single-strand breaks are repaired?. If the T4 DNA ligase function is missing, singleand double-strand breaks accumulate in replicating DNA. Under these circumstances late-transcription-competent DNA should be preserved after the arrest of replication. Our experiments clearly indicate that in order for even partial preservation of competent template to occur, a gene 46 mutation is also essential. The reason for this appears to be that unligated DNA is subject to rapid degradation under the control of gene 46. Exonucleolytic action probably starts at the single-strand breaks and destroys binding/initiation sites for late transcription. We do not know whether gene 46 mutations are the best or the only way of interfering with this degradation. The possibilit,ies for incisive biochemical or genetic manipulation of nucleases in T4 development are currently limited. Accordingly, the intrinsic involvement of the gene 46 product in late transcription-replication coupling is not yet specified in our model. On the other hand, we have shown that other-than-newly replicated DNA can become a competent template for late transcription: upon inactivation of DNA polymerase and ligase, previously synthesized DNA of the triple mutant tsP36 tsA80 amNl30 (genes 43, 30, 46) can sustain some late messenger synthesis (Fig. 5). In fact, some late transcription occurs in cells infected with these mutants in the absence of any viral replication. We have shown in the preceding paper that the proteins necessary for late transcription can be synthesized in the absence of DNA replication, therefore the presence of late functions directly reflects DNA competence. At the time when this late transcription starts, parental DNA has suffered extensive single-strand, but very little double-strand, breakage (Fig. 9 and Cascino et al., 1970). Therefore we propose that DNA competence can occur as the result of endonuclease (endonuclease L) action without replication; the lifetime of competent DNA is limited by ligation and also, in some circumstances at least, by exonuclease action. If competence can result from endonuclease L action, why is there any replication coupling of late transcription Z Presumably, for one of these reasons : (1) only a small fraction of control sites is created by endonuclease L action or (2) endonuclease L works much more poorly on mature than on newly synthesized DNA. In fact, there is no assurance that the residual replication-uncoupled late transcription depends on endonuclease L action although this seems an attractive hypothesis since it points to the analogy between residual, replication-uncoupled late transcription and replicationindependent late gene expression at very high multiplicities of infection. In this connection, Hosoda $ Mathews (1970) report a particularly interesting observation about T4 DNA fragments. They have found that 16 s DNA fragments which they suppose to arise from endonuclease action rather than from the primary discontinuous replication process, hybridize preferentially to the T4 DNA r-strand. They have not found such a bias in the hybridization of smaller (8 S) or larger DNA fragments. This unequal distribution of interruptions among T4 DNA r and 1 strands could be related to the predominantly r polarity of T4 late RNA. BBguin (manuscript in preparation) has independently come to the conclusion that DNA interruptions are essential for T4 late transcription. In closing, we mention two possibilities about late transcription that are suggested by these experiments. First, it seems possible that the maturation of competent DNA t Strictly speaking, absenoe of replication

some single-strand breaks are either not repaired but their abundance is presumably much lower.

or are reintroduced

in the

T4 REPLICATION-TRANSCRIPTION

UNCOUPLING

119

may regenerate active late transcription enzyme. Transcription is, of course, cyclic and the proper leaving mechanisms are an essential element of a metabolically stable set of transcription effecters. DNA ligation could be the leaving mechanism for initiation or polymerization components of late transcription. In that sense, replioationlate transcription coupling would have to be regarded as a particular and specific mechanism of transcription ancl not as a general regulatory mechanism of virus production. Second, if late transcription and replication take place in relatively close proximity, there may be interactions between the proteins of late transcription and of the replication complex. Studies of these questions are now in progress. This research was supported by research grant GM16880 awarded by the U.S. Public Health Service and by an award from the Life Insurance Medical Research Foundation. One of us (E.P.G.) holds a Research Career Development Award. We are grateful to N. Cozzarelli and J. Hosoda for helpful comments and discussions, to J. S. Wiberg for comments on the manuscript, and to F. Beguin for communicating his findings prior to publication. REFERENCES Bernstein, H. (1968). Cold Spr. Harb. Symp. Quunt. Biol. 33, 325. Belle, A., Epstein, R. H., Salser, W. & Geiduschek, E. P. (1968). J. Mol. Biol. 33, 339. Brody, E. N. & Geiduschek, E. P. (1970). Biochemistry, 9, 1300. Cascino, A., Riva, S. & Geiduschek, E. P. (1970). Cold Spr. Harb. Symp. Quant. Biol. 35, in the press. Chan, V. L., Shugar, S. & Ebisuzaki, K. (1970). Virology, 40, 403. Epstein, R. H., Belle, A., Steinberg, C. H., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Susman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Spr. Ha&. Symp. Quant. Biol. 28, 375. Frankel, F. R. (1966). J. Mol. Biol. 18, 127. Frankel, F. R. (1968). Proc. Nat. Acud. Sci., Wash. 59, 131. Gefter, M., Hausmann, R., Gold, M. & Hurwitz, J. (1966). J. BioZ. Chem. 241, 1995. Geiduschek, E. P. & Grau, 0. (1970). In RNA Polymers-se and Transcription, ed. by L. Silvestri, p. 170. Amsterdam: North Holland Publishing. Gulls, A., Salser, W. & Szybalski, W. (1968). Fed. Proc. 27, 646. Hosoda, J. (1967). Biochem. Biophya. Res. Comm. 33, 670. Hosoda, J. k Levinthal, C. (1968). F$roZogy, 34, 709. Hosoda, J. & Mathews, E. (1968). Proc. Nat. Acud. Sci., Wash. 61, 997. Hosoda, J. & Mathews, E. (1970). J. Mot. Biol. 54, in the press. Karam, J. 13. (1969). Biochem. Biophys. Res. Comm. 37, 416. Kozinski, A. W. (1968). Cold Spr. Harb. Symp. Quant. Biol. 33, 376. Kozinski, A. W. & Mitchell, M. (1969). J. Virology, 4, 823. Kutter, E. M. & Wiberg, J. S. (1968). J. Mol. Biol. 38, 395. Mark, K. K. (1965). Ph.D. Thesis, University of Oregon. Newman, 5. & Hanawalt, P. (1968). J. Mol. Biol. 35, 639. Okazaki, R., Okazaki, T., Sakabe, K. & Sugimoto, A. (1967). Japan J. Med. Sci. Biol. 20, 255. Okazaki, R., Okazaki, T., Sakabe, K., Sugimoto, K. & Sugino, A. (1968). Proc. Nat. Acad. Sci., Wash. 59, 598. Pulitzer, J. F. (1970). J. Mol. Biol. 49, 473. Richardson, C. C., Masamune, Y., Live, T. R., Jacquemin-Sablon, A., Weiss, B. & Famed, G. C. (1968). Cold Spr. Harb. Symp. Quant. Biol. 33, 151. Riva, S., Cascino, A. & Geiduschek, E. P. (1970). J. Mol. Biol. 54, 85. Wiberg, J. S. (1966). Proc. Nat. Acad. Sci., Wash. 55, 614.