- Email: [email protected]
Host and phage-coded functions required for coliphage N4 DNA replication
VIROLOGY
150,33-44 (1986)
Host and Phage-Coded Functions Required for Coliphage N4 DNA Replication D. GUINTA,’
J. STAMBOULY: S. C. FALCO: L. B. ROTHMAN-DENES4
J. K. RIST,
AND
Departmenta of Molecular Ba,
Genetics and Cell Biologzl and of Biochemistry and Molecular The University of Chicago, Chicago, Illinois 60637
Received October 10, 1985; acwpted Now&e-r
27, 1985
Escherichia coli strains containing mutations in various deoxyribonucleic acid synthesis cistrons have been tested for their ability to support bacteriophage N4 growth and, specifically, N4 DNA synthesis. N4 DNA synthesis is independent of the activity of the products of the E. coli dnaA, dnaB, dnaC, dnaE, dnaG, and rep genes. In contrast, N4 DNA replication requires the products of the dnaF, (ribonucleotide reductase) and lig (DNA ligase) genes of E. co& N4 DNA replication, specifically processing of short DNA fragments requires the 5-3 exonuclease activity of the po&f gene product. However, its DNA polymerizing activity is not required. In addition, the sensitivity of N4 DNA synthesis to inhibitors or temperature-sensitive mutants of E, coZi DNA gyrase suggests that this activity is required for N4 DNA synthesis. To date, we have found five N4 gene products required for N4 DNA replication: d&n (a single-stranded DNA binding protein), dnp (a DNA polymerase), dn.s (unknown function), vRNAp (the N4 virion-associated, DNA-dependent RNA polymerase) and ezro (a 5’-3’ exonuclease) 0 19% Academic PWS. Inc. INTRODUCTION
host DNA-dependent RNA polymerase (Rothman-Denes and Schito 1974; Zivin et a& 1981). Early RNAs appear in cells pretreated with chloramphenicol and rifampicin, indicating that neither an active E. coZi RNA polymerase nor translation of the phage genome is required for early RNA synthesis. The chloramphenicoland rifampicin-resistant transcription is due to a virion-encapsulated, DNA-dependent RNA polymerase (Falco et aL, 1977). In this paper we show that this RNA polymerase is directly required for N4 DNA replication as well. Moreover, in order to understand how N4 DNA synthesis is controlled, we have studied the host and phage requirements for N4 DNA synthesis.
N4 is a lytic bacteriophage that infects coli K 12 strains. Its virions contain 72 kb of linear double-stranded DNA with 400 to 450 bp of direct terminal repeats (Zivin et al, 1980). The structure of the left (or early) end of the genome is unique containing a 7-base 3’ overhang (L. Haynes, unpublished). The right end is variable; there are at least five different ends differing from each other by 10 bp giving rise to variability in the length of the terminal repeats (H. Ohmori and L. Haynes, unpublished). N4 differs from other DNA-containing bacteriophages in that synthesis of its early and middle RNAs does not require the activity of the Escherichia
MATERIALS
1 Present address: Department of Genetics, Stanford University School of Medicine, Stanford, Calif. ’ Present address: University of Pittsburgh, Pittsburgh, Pa. a Present address: E. I. duPont de Nemours Central Research, Wilmington, Del. ‘To whom requests for reprints should be addressed.
AND
METHODS
Baderial and phage strains. E. coli strain CR34 and its derivatives X9258 (dnaA46), JW184 (dnaB43), E486 (dmE486) were obtained from J. Wechsler (Wechsler and Gross, 1971). The rep 3 derivative of CR 34, NT rep 3, was provided by M. Gellert (Den33
004%6822/86 $3.00 Copyright All rights
8 19% hy Academic Press, Inc. of reproduction in any form reserved.
34
GUINTA
hardt et aL, 1967). M21 (DllO polA endol) and its dnuF El01 derivative, isolated by J. Wechsler (Wechsler and Gross, 1971) were obtained from R. Moses. DG 76 and its derivatives PC2 (dnaC2) and PC3 (dnuG3) were provided by P. Carl (Carl, 1970). H560 and its coumermycin-resistant derivative was constructed and provided by C. Peebles (Falco et a& 1978). KS463 and RS5064, its polAex1 derivative, were obtained from B. J. Bachmann, E. co,?iGenetic Stock Center, Yale University (Konrad and Lehman, 1974). GR523 and its Zigts251 derivative, GR501 were kindly provided by R. Sternglanz (Dermody et aL, 1979). N4 am12 was isolated by G. C. Schito. N4 33 am7, N4 am257, and N4 ts150 (Falco et al, 1977) were isolated by hydroxylamine mutagenesis in this laboratory. Media. M9 minimal salts medium (Bolle et aL, 1968), containing 0.4% glucose, 0.2% casaminoacids, 5 pg/ml thiamine, and 20 @g/ml thymine was used in all experiments. Chemicals. Coumermycin was a gift of Dr. N. C. Cozzarelli. [methyl-3H]Thymidine (50-62 Ci/mmol) was purchased from Schwarz-Mann. Lj3H]Aminoacid mix (1 mCi/ml) and En3Hance were purchased from New England Nuclear Company. Burst measurements. E. coli strains were grown at the permissive temperature in M9 minimal medium to a density of approximately 5 X lo* cells/ml (ODsm = 0.5). Cells were centrifuged and resuspended in fresh M9 medium prior to infection. The resuspended cells were divided in half, one-half of the cells were placed at 33” and the other half was placed at 43”. Parental strains and strains with immediate-stop dna ts mutations (dnuB, dnuE, dnuF) were preincubated at the restrictive temperature for 15 min, while the delayed-stop dnu strains were preincubated at the restrictive temperature for 60 min. After this period, wild type N4 phages were added to the cultures at a multiplicity of infection of 10. Unadsorbed phage were inactivated with N4 specific antiserum (final K = 10) added 5 min postinfection. The cells were incubated for an additional 5 min and then an aliquot of each infected culture was diluted 105fold into warmed M9 minimal media. At
ET AL.
this time, an aliquot was immediately plated E. coli W3350 to determine the number of infectious centers. The diluted cultures were incubated a total of 3 hr, after which samples were removed and the cells lysed with chloroform. Burst size was determined by titering the lysate on w3350. Measurement of the rate of DNA suntheti. Cells were grown and resuspended in M9 minimal media as described above. In the case of dnu ts mutants, thymine was added to a final concentration of 100 pg/ ml. After preincubating and treating the cells as above, N4+ was added at a multiplicity of 10. At various times after infection, 0.2-ml samples were removed and incubated for 2 min with 5 &i of [‘HIthymidine. At the end of the pulse, 0.05-ml samples were treated as described previously (Rothman-Denes and Schito, 1974). Alkaline sucrose gradients. Cells were infected and gradients run according to the method of Okazaki et al. with monitor modifications (Okazaki et uL, 1978). E. coli was grown to an ODszoof 0.5 in M9 media at 33” and infected with phage at an m.o.i. of 10. Thirty-six mintues after infection, the culture was shifted to 43” and pulsed 4 min later with 10 &i [3H]thymidine/ml of culture for lo- or 30-set intervals. In pulse and chase experiments, cells were pulsed for 10 set or 1 min, followed by a 4min chase with 2 mg/ml of unlabeled thymidine. Samples were removed and lysed with alkali according to Masamune et al. (1971). Cells (0.1 ml) were added to 0.1 ml ice cold 10 N NaOH, 0.5 1MEDTA, 0.1 M NaCN. After a lo-min incubation on ice, 0.1 ml 20% Sarkosyl NL97 was added along with [14CjN4DNA as a marker. Each sample was layered on a prechilled 9.0 ml linear 5-20s w/v sucrose gradient overlaying a 1.0 ml 80% w/v sucrose cushion. Sucrose solutions contained 0.1 M NaOH, 0.9 M NaCl, and 1 m&f EDTA. Gradients were centrifuged at 4” for 16 hr at 26,000 rpm in an SW41 rotor. Gradients were collected in 11 drop fractions from the bottom of the tube and acid-insoluble radioactivity was measured. Polyucrylumide gel electrophoresis and uutorudiogruphy. E. coli W3350 cells were
COLIPHAGE
N4 DNA
treated with rifampicin (200 rg/ml) 20 min before infection. Two minutes after infection with the indicated phages, L-[~H]aminoacid mix was added to 50 &i/ml. Infected cells were incubated for 60 min, collected by centrifugation, resuspended, and eleetrophoresed on a 15% polyacrylamide gel as described (Falco and RothmanDenes, 1979). The gel was prepared for fluorography by treatment with En’Hance according to manufacturer’s instructions. Extraction of labeled intracellular DNA and DNA-DNA h&ridimtim One-milliliter samples were treated according to Botstein (1968) except that after dialysis, RNAse was added to 0.1 mg/ml and incubated for 5 min at 37’ followed by 3 min at 70”. Sarkosyl was then added to 2% and the incubation was continued for 20 min at 70’ followed by 1 hr at 37”. Pronase was added to a final concentration of 2 mg/ml. The samples were incubated for 3 hr at 37” and dialyzed overnight against 10 m&Z Tris-HCl pH 8,10 mMEDTA, 0.15 MNaCI. DNA filters containing 10 pg of E. coli DNA or 20 gg of N4 DNA were prepared according to Gordon and Rabinowitz (1973). The labeled DNA was denatured with alkali. Hybridization was carried out in a total volume of 1 ml containing 1X SSC, 1% SDS at 65” for 14 hr. RESULTS
Production upon Ir@ectim of E. coli dnats Mutants under Restrictive cditim
N4 Progeny
To study the requirement of E. coli dna gene products for N4 growth, E. u& strains carrying temperature-sensitive mutations in various dna genes were tested at the restrictive temperature for their ability to support N4 growth. Table 1 shows the burst size of N4 upon infection of these strains as plaque-forming units per infective center. For dnaA, dnaB, and dnaG mutants, more progeny were made at 43O than at 33’. For the dnaE and dnaC mutants a large number of phage were released at 43”, although the burst size was smaller. The polh mutation, which affects the DNA polymerase activity of the protein, or rep,
35
REPLICATION TABLE
1
N4 BURST SIZE IN E. wli dm” STRAINS Burst/ infective center Relevant genotype
Strain
Ratio 43”/33”
33O
43”
377 614 583 752
1174 745 1409 289 1625
3.11 1.21 2.42 0.38 -
546
463
CR34
Wild type
X9258 JW184 E486 NT516
dwz.446 dnaB43 dnaE486 (polQ rep 3
DG76 PC2 PC3
Wild type
dnaCi? dmG3
34
22
178
261
0.85 0.65 1.47
M21 ME431
PdAl pdA1, d&El01
943 148
905 0
0.96 0.00
KS463 RS5064
Wild type pdAt?Xl
400
1800 30
0.07
GR523 GR501
ligts251
1333 1200
1842 38
1.38 0.03
Wild type
a Conditions
-
-
as under Materials
-
and Methods.
which affects the helicase II activity, did not affect N4 growth. In contrast, infection of the E. coli dnuF and lig mutants did not yield any progeny phage at 43”. It therefore appears that N4 phage development does not absolutely require the activity of the gene products of the host genes dnaA, dnuB, dnaC, dnaE, and dnaG, and the DNA polymerase activity of the polA gene product. The activities of the E. coli enzymes ribonucleotide reductase and DNA ligase are, however, required. N4 DNA Sgnthesis in E. coli Mutant Hosts To examine the effects of the above mutations directly on N4 DNA synthesis, infected cells were pulse-labeled with r3H]thymidine. Immediately after N4 infection of E. coli CR34, [‘Hlthymidine incorporation decreased due to cessation of host DNA synthesis (Fig. IA). This was followed by a burst of incorporation due to N4 DNA synthesis (Fig. 1A). Pretreatment of cells with chloramphenicol prevented
36
GUINTA
ET AL.
TIME
(min)
FIG. 1. Rate of [‘Hlthymidine incorporation after N4 infection of wild type and temperaturesensitive DNA synthesis mutants of E. co& at 43”. Conditions and strains as under Materials and Methods. (A) 0, wild type; 0, dnuAts; A, debts; n , dnuEts. (B) 0, wild type; 0, dnacts; A, dnaGts.
both the termination of host DNA synthesis as well as the initiation of N4 DNA synthesis, suggesting that both processes require phage protein synthesis (RothmanDenes and Schito, 1974). N4 DNA synthesis occurred normally when dnaA, drmB, and dnaE mutants were infected at 43” (Fig. lA), where the pertinent proteins were no longer functional in host DNA synthesis. Figure 1B shows the profile of rH]thymidine incorporation after N4 infection of the dnaC and dnaG temperature-sensitive derivatives of E. coli DG’76. Again, despite the absence of the respective functional dna gene products, N4 DNA contin-
TIME
FIG. 2. Rate of [8H]thymidine mutants E. coZi under restrictive (B) 0, kg+; 0, Zigts.
ues to be synthesized at the restrictive temperature. Similar results (not shown) are obtained upon infection of E. coli cells carrying rep mutations. Figure 2A shows the pattern of incorporation for mutants deficient in the polymerizing activity of DNA polymerase I and in ribonucleotide reductase. In strain M21, carrying the @Al mutation, significant levels of DNA synthesis occurred at both 33” (not shown) and 43”. However, the ME431 derivative of M21, which bears a mutation in dnaF, failed to support N4 DNA synthesis at 43”. Similar results were obtained when a strain of E. coli carrying
(min)
incorporation after N4 infection of &Al, dmF (A) and Zig (B) conditions. Infections were at 43“ (A) 0, @Al; 0, por!Al dn&ts.
COLIPHAGE
N4 DNA REPLICATION
37
a temperature-sensitive mutation in DNA ligase, &ts251, was infected at the nonpermissive temperature (Fig. 2B). These data therefore demonstrate that N4 DNA synthesis does not require the host dnaA, dnaB, dnaC, dnaE, dnaG, rep, and polA functions but requires functional ribonucleotide reductase and DNA ligase. Involvement of the 5’4 Ex~ucleolytic Activitg of DNA Polgwase I in N,$DNA &@thesis As shown above, the DNA polymerizing activity of polA is not required for N4 growth. However, no N4 was produced upon infection of E. coli cells deficient in the 5’3’ exonuclease activity of mL4 (Table 1). This result was observed in polAexl cells, which have a temperature-sensitive, conditional lethal mutation in the DNA polymerase I 5’-3’ exonuclease, as well as in polA12 cells which are defective in coordinating the polymerase and 5’-3’ exonuclease activities of Pol I. The pattern of r3H]thymidine incorporation after N4 infection of these mutant strains, however, was norma (not shown). In order to explain this discrepancy, we analyzed the accumulation of N4 Okazaki fragments in wild type or exonuclease mu-
IO
20
10
30
20
JO
i=RAcTulN
FIG. 3. Effect of E. coZipdrlez1 mutation on N4 DNA synthesis. (A) E. wli KS463, (B) E. co& RS5064, a polAex1 derivative of KS463. Cells were infected at 43” with wild type N4 and intracellular DNA was labeled for 10 set (0) or 30 see (0). Cells were lysed and alkaline sucrose gradients were run as described under Materials and Methods. Virion length N4 DNA was found in fractions 1-3 in all gradients. Sedimentation is from right to left.
FIG. 4. Pulse and chase of labeled N4 DNA in polAex1 cells. E. coli RS5064 was infected with wild type N4 at 43”. Cells were labeled with [BHjthymidine for 10 set (A) or 1 min (O), followed by a I-min chase with an excess of unlabeled thymidine (0). Cells were lysed and alkaline sucrose gradients were run as under Materials and Methods. Virion length DNA was found in fractions 1-3. Sedimentation was from right to left.
tant infected cells, since the 5’-3’ exonuclease activity of polA is involved in that process in other phage systems. The results of this experiment are shown in Fig. 3. Small DNA fragments did not accumulate in the wild type parental strain. However, they did accumulate in both the polAex1 and polAl2 (not shown) mutants under restrictive conditions. Figure 4 shows that these small fragments accumulating in polAex1 cells infected with N4 could be chased into larger DNA, demonstrating the slow conversion of nascent DNA into mature DNA rather than a slow process of degradation. This suggests that these small fragments are indeed Okazaki fragments. The 5’-3 exonuclease of the host DNA polymerase I is, therefore, required to process N4 Okazaki fragments. Efect of DNA Gyrase Inhibitors DNA Synthesis
on N4
N4 early RNA synthesis, which is required for all subsequent events during N4 infection, requires active E. coli DNA gyrase (Falco et al, 1978). In order to assess the possible involvement of E. co2i DNA gyrase in N4 DNA synthesis, gyrase inhibitors were added after the onset of DNA replication (25 min postinfection). Coumermycin is a strong inhibitor of N4
33
GUINTA
ET AL.
DNA synthesis. Maximal inhibition was achieved within 5 min of the addition of the drug (Fig. 5). The effect was not observed in coumermycin-resistant infected cells, suggesting that the effect is through inhibition of E. coli DNA gyrase, and that its activity is required for N4 DNA replication. Similar results were obtained after shift-up to the restrictive temperature of infected cells carrying temperature sensitive mutations in the ggrA locus (not shown). Involvement of N&%ded DNA Replication
Functions
in TIME
(min)
FIG. 6. Rate of mjthymidine incorporation after At present, suppressor-sensitive mutants of N4 have been isolated in eight cis- N4 mutant infection of E. cd W3350. (A) 0, N4+; n , trons which are required for DNA repli- um15; A, am.23 0, am126. (B) 0, awt257; A, am33A7; cation. Mutations in one cistron, coding for l , am12. Other conditions as under Materials and Methods. a 5’-3’ exonuclease, show a temperaturedependent DNA arrest phenotype and will not be discussed further here (D. Guinta, alleles am126, am15, and am23, are reS. Spellman, and L. Rothman-Denes, un- quired for N4 middle RNA synthesis published). Mutations in the seven other (Rothman-Denes and Schito, 1974; Zivin et cistrons express a DNA negative pheno- c& 1981; Zehring et al, 1983). Results of type, i.e., no N4 DNA synthesis is detected experiments with temperature-sensitive (Fig. 6A). Three of these cistrons, rnaII a, mutants in these three latter cistrons inrnaII b, and rn.uII c, represented by mutant dicate that their gene products are not directly involved in N4 DNA replication since inactivation of the product, after middle RNA synthesis has occurred, has no effect on N4 DNA replication (not shown). The products of three other N4 cistrons, dnp (a DNA polymerase), &n (DNA binding protein), and dns (unknown function), represented by mutants am257, am33A7, and am12, are directly involved in DNA replication (Fig. 6B) (Schito, 1973). The products of two of these genes can be easily identified on polyacrylamide gels when the pattern of proteins labeled after infection of suppressor and nonsuppressor containing strains are compared (Fig. 7). The product of the dns cistron is probably a 1 1 protein of 78,000 mol wt (~2) since it is 40 so 10 20 JO missing in am12 infected cells. An abunTIME (min) dant protein, (~8: 30,000 mol wt) is missing FIG. 5. Effect of coumermycin on N4 DNA synthesis. after awz33A7infection, suggesting that it Two-minute pulses of @IJthymidine were performed is the product of the dnp cistron. No difas described under Materials and Methods. Coumerferences could be detected after am257 inmycin (20 g/ml) was added at 25 min after infection of E. coZiH566 mall (A) or E. cm.5 H566 m&l CaL’ (B). fection. This is not due to the particular n , no coumermycin; 0, coumermycin. mutant infection used since similar results
COLIPHAGE
N4 DNA REPLICATION
39
polymerase is resistant (Falco et aL, 1977). Further characterization of N4ts150 revealed that this enzyme pIays a second role in N4 development. The effect of raising the temperature on the rate of DNA synthesis after N4 wild type and N4ts150 infection is compared in Fig. 8. When E. coli cells were infected with wild type N4 at 33”, pulse-labeling with [SH)thymidine yielded a characteristic pattern shown in Fig. 8A. A similar pattern was obtained upon infection at 43”. When cells infected at 33” were shifted to 43” at 25 min after t ~8 infection, no change in the pattern of thymidine incorporation was observed. Infection with N4ts150 yielded strikingly different results (Fig. 8B). At the permissive temperature the pattern was similar to that observed after N4 wild type infection. In contrast, at 43”, a slow decline in the rate of thymidine incorporation was seen. The rapid increase in rate, characteristic of the onset of viral DNA replication, did not occur. This result was expected because the virion RNA polymerase, which am u N4 is inactive at 43“, is required for N4 early 257 T2” &ii RNA synthesis and therefore for all postFIG. 7. N4 induced proteins after wild type and mu- infection protein synthesis. When cells intant infection. Conditions as under Materials and fected at 33” were shifted to 43” at 25 min Methods. U: uninfected control. PO: indicates place of after infection, the rate of thymidine inmigration of a protein which has been identified as corporation rapidly decreased to less than the dnp gene product (K. Rist, unpublished). P2: pro10% of the level attained at 33”. tein missing in dns mutant (am12) infection. P8: proThe effect of temperature shift on DNA tein missing in &p mutant (aWA7) infection). synthesis in N4ts150 infected cells was not indirectly due to the inhibition of synthesis were obtained with other dnp mutants, of a protein required for DNA replication suggesting that the product of dnp is either or maturation. Addition of chloramphentoo scarce to be detected by this technique icol at 25 min after infection to a wild type or it comigrates with other phage or cel- N4 or N4ts150 infected culture did not inhibit N4 DNA synthesis, although an inlular proteins. hibition of the increase in the rate of DNA replication was observed. Requirement of the N4 virion-encapsulated The cessation of rH]thymidine incorRNA polgmerase in N4 DNA replicaporation, which followed a shift to the retion strictive temperature in N4ts150 infected N4ts150 is a temperature-sensitive mu- cells, was specific for N4 DNA synthesis. tant of N4 which shows temperature sen- Table 2 shows the results of hybridization sitive early N4 RNA synthesis (Falco et al, of DNA extracted from wild type N4 or 1977). We have shown that this mutation N4ts150 cells to either E. co&ior N4 DNA. resides in the N4 virion-encapsulated RNA DNA labeled from 15 to 20 min, or 35 to 40 polymerase since the enzyme purified from min after N4 or N4ts150 infection at 36”, N4ts150 virions is sensitive to thermal in- hybridized exclusively to N4 DNA. When activation while wild type N4 virion RNA the temperature was shifted from 36 to 43”
40
GUINTA
ET AL.
TIME AFTER INFECTION
bth)
FIG. 8. Rate of [%I]thymidine incorporation in wild type or ts150 N4 infected cells. l , infection at 33’; 0, infection at 43’; A, infection at 33’, chloramphenicol was added at the time indicated by the arrow; (x, infection at 33”, temperature shifted to 43” at the time indicated by the arrow. Other conditions as described under Materials and Methods.
at 21 min after wild type N4 infection and the DNA was labeled from 35 to 40 min, the DNA that was synthesized hybridized to only N4 DNA. In contrast, when the temperature was shifted during N4ts150 infection, very little label was incorporated into DNA during the pulse and none of it hybridized to N4 DNA. Genetic evidence indicates that the double phenotype exhibited by N4ts150-i.e., temperature-sensitive early RNA synthesis and DNA replication-is due to a single mutation. It has not been possible to separate the two phenotypes by back-crossing N4ts150 to wild type N4. Reversion of both phenotypes occurs simultaneously at a frequency of 3 X 10e5.Furthermore, a second independently isolated N4 mutant, N4ts245, has the same double phenotype. Reversion of both phenotypes in N4ts245 occurs simultaneously at a frequency of 2 X 10e4 (data not shown). Genetic recombination indicates that N4ts150 and N4ts2.45are two different but closely linked mutations (data not shown). We therefore conclude that the N4 virion RNA polymerase plays a direct role in N4 DNA replication and mutations in it define a new cistron with a DNA negative phenotype.
DISCUSSION
The large N4 genome is unusual because of the structure of its termini. The virion DNA is ‘72kb in length and contains direct terminal repeats of 400-450 bp (Zivin et aL, 1980). The genetic left end of the genome (early region) is unique and contains a ‘7 base single-stranded 3’ end (3’CCATAAA) (Ohmori and Haynes, unpublished). The TABLE 2 ANALYSIS OF HYBRIDIZATION
DNA
SYNTHESIS BY DNA-DNA IN N4 INFECCED E. wli CELLS cpm hybridized to
Temperature 36’ 36’
43Ob
Labeling period (min)
Phage
Input cpm
-5-o 15-20 15-20 35-40 35-40 35-40 35-40
N4+ ts150 N4+ te.150 N4+ ts150
1341 1582 1414 1704 1394 202.3 147
N4 DNA’ 5 1073 666 1556 912 1252 0
E. wli DNA” 2.22 15 13 4 18 30 34
a Blanks, 24-56 cpm, were subtracted. ’ 36’C infected cultures were shifted to 43’ at 21 min after infection. Other conditions as under Materials and Methods.
COLIPHAGE
N4 DNA
right end is not unique; one major and four minor ends exist in the population, which differ by 10 bp over a 50-nucleotide-long region. These ends also have 3’ singlestranded overhangs (Ohmori and Haynes, unpublished). Although 3’ tails are common as a result of restriction endonuclease (Roberts, 1985) or topoisomerase cleavage (Reed and Grindley, 1981), they are not found at the ends of other phage and viral genomes. N4 shares its unusual terminal structure with the macronuclear DNAs of hypotrichous ciliates, such as Oxgtricha nova which has 16 base 3’ single-stranded tails of the sequence G4T4G4T4(Klobutcher et al, 1981). In order to understand how N4 DNA replicates and generates its 3’ singlestranded ends, we have determined those E. coli functions which are required for N4 DNA replication as well as whether N4 codes for any replication functions of its own. By analysis of phage burst size and measurement of N4 DNA synthesis we have shown that N4 does not require the activity of the host dnuA, dnaB, dnaC, dnaE, dnuG, rep, or polA gene products. The lack of a requirement for either of the host DNA polymerases III or I (dnaE or polA) strongly suggests that N4 codes for its own DNA polymerase. The independence from dnaG, dnuB and dnuC products implies that N4 provides its own priming machinery as well. Finally, it is not surprising that the dnaA product is not required for N4 replication since specific replicons should be activated by specific initiation proteins (Jacob and Brenner, 1963). With the use of conditional-lethal mutants, five N4 genes have been shown to be required for N4 DNA synthesis thus far. Mutants in three genes, dns, dnp, and dbp, show a DO phenotype; that is, no N4 DNA synthesis is detectable. The function of the dnp and @genes has been determined using an in vitro N4 DNA replication system. While extracts made from wild-type N4 infected E. coli support specific replication of N4 DNA, extracts made from N4 mutants in the dnp or dbp genes do not. This has allowed the purification of the products of the dnp and dbp genes from wild type
REPLICATION
41
N4 extracts by complementation of the appropriate mutant extract (Rist et al, 1983). The purified proteins have been shown to be a DNA polymerase (drip) and DNA binding protein (d&n) (Rist et d, submitted). The product of the dns gene, although identified on SDS-polyacrylamide gels (Fig. 7), has not been purified, and its function is not known. Two other N4-coded functions required for N4 replication have been identified but the elucidation of their roles awaits an understanding of the mechanism of N4 DNA replication. The N4 exo gene codes for a 5’3’ exonuclease which can be purified by in vitro complementation for N4 DNA replication of an exo mutant-infected cell extract. Only one mutant allele of this gene has been isolated and it has a temperaturedependent DNA arrest phenotype (D. Guinta, S. Spellman, and L. B. RothmanDenes, unpublished). Analysis of the role of the N4 virion RNA polymerase in replication is complicated by its involvement in all early phage events. When this aspect of the virion polymerase’s function is separated from its effect on DNA synthesis by temperature shift-up experiments, it is clear that the polymerase is directly required for N4 replication. The virion RNA polymerase has been shown to accurately initiate transcription on a denatured N4 DNA template in vitro (Falco et aL, 1978; Haynes and Rothman-Denes, 1985). This enzyme will also accurately transcribe double-stranded DNA in vitro if the DNA carries a promoter and is supercoiled and E. cola’single-stranded DNA binding protein (ssb) is present (P. Markiewicz, J. Chase, unpublished). In tivo, transcription by this enzyme requires the activity of E. co& DNA gyrase (Falco et aZ., 1978), also suggesting a requirement for unwinding of the helix in the order for the polymerase to recognize its promoters. It is not yet known what replicative form of N4 DNA is the substrate for the virion polymerase in N4 replication, nor is it known whether this RNA polymerase is required for priming at the N4 origin of replication or for some step in possible lagging strand synthesis.
42
GUINTA
In general, when a virulent bacteriophage supplies its own DNA polymerase, it also supplies many of the proteins required to maintain a moving replication fork. The T7 replication fork uses only three phage-encoded proteins to maintain leading-strand synthesis and prime and maintain lagging-strand synthesis (Richardson, 1983). The priming (Hillebrand et al, 1979; Roman0 and Richardson, 1979) and helicase (Lechner and Richardson, 1983; Matson et aL, 1983) activities reside in a single, multifunctional protein, the gene 4 product (Kolodner et ah, 1978). The single-stranded DNA binding protein (Reuben and Gefter, 1974) is the product of gene 2.5 (Dunn and Studier, 1981). Its activity is dispensible since it can be substituted by the host DNA binding protein (Araki and Ogawa, 1981). The DNA polymerase activity resides in a complex composed of a phage-coded polypeptide, the gene 5 product, and E. coli thioredoxin (Modrich and Richardson, 1975). In contrast, the bacteriophage T4 replication fork requires the activity of seven phage-coded proteins. The T4 DNA polymerase (Goulian et ah, 1968) and singlestranded DNA binding (Alberts and Frey, 1970) proteins are both single polypeptides. However, three “accessory” proteins, the products of genes 45,44, and 62 (Barry and Alberts, 1972; Nossal, 1979), are required by T4 DNA polymerase for efficient polymerization. These proteins act as a “clamp” for T4 DNA polymerase, increasing the rate of polymerization and eliminating pausing in an ATP-dependent fashion (Alberts et ak, 1980; Huang et aL, 1981; Mace and Alberts, 1984). The T4 primase activity requires the combined action of the T4 genes 41 and 61 protein products (Morris et aL, 1979; Silver and Nossal, 1978, 1982; Liu and Alberts, 1981). Helicase activity is present in the 41 protein (Venkatesan et al, 1982). The N4 genome is large enough to code for most of its replication functions. We have identified two proteins required for maintenance of an active replication fork, a DNA polymerase and a single-stranded DNA binding protein. N4 probably encodes
ET AL.
its own primase and helicase as well. It will be interesting to see if these activities reside in replication genes that we have already identified or in, as yet, unidentified genes. Furthermore, as we have no mutant alleles in N4 recombination genes, we cannot assess the contribution, if any, of recombination to N4 DNA replication. Four host gene products are required for N4 DNA synthesis: DNA gyrase, ribonucleotide reductase, DNA ligase, and the 5’3’ exonuclease function of DNA polymerase I. The requirement of the host DNA gyrase for N4 replication was shown by the addition of the specific DNA gyrase inhibitors after the onset of N4 DNA replication. Because the N4 virion RNA polymerase requires the activity of DNA gyrase for transcription of the early region of the N4 genome in viva (Falco et cd, 1978), it is possible that DNA gyrase is also required to aid the virion polymerase in its replication function as well. However, DNA gyrase may be required directly for N4 DNA synthesis. T4 genes 39, 52, and 60, which encode proteins that form a type II topoisomerase, have mutant phenotypes characterized by a reduction in the rate of the initial phase of T4 DNA replication (reviewed in Kreuzer and Huang, 1983; Marians, 1984). T4 topoisomerase mutants are predominantly defective in initiation of replication through recombinational intermediates (G. Mosig, personal communication). The N4 requirement for ribonucleotide reductase probably serves to provide a pool of nucleotides for N4 DNA synthesis. N4 shuts off host DNA synthesis, but does not degrade the host genome (Rothman-Denes and Schito, 1974; see also Fig. 1). The requirements for the host ligase and DNA polymerase I 5’-3’ exonuclease, in conjunction with the demonstration of short nascent DNA in N4 infected cells, suggest that the mechanism of N4 replication includes some form of discontinuous DNA synthesis. These nascent DNA chains have a size similar to that of T7 Okazaki fragments (Okazaki et al, 1978) (1-6 kb) and can be chased into longer DNA molecules (M. S. Pearle and D. Guinta, unpub-
COLIPHAGE
N4 DNA REPLICATION
43
DNA to the beginning of gene 4. J. MoL BioL 148, 303-330. FAL.CO,S. C., and ROTHMAN-DENES,L. B. (1979). Bacteriophage Nd-induced transcribing activities in Eschmichia c&i I. Detection and characterization in cell extracts. Vi* 95.454-465. FALCO,S. C., VANDERLAAN, K., and ROTHYAN-DENES, L. B. (1977). Virion-associated RNA polymerase required for N4 development. Proc NatL AuuL Sci USA 74.520-52.X FALCO, S. C., ZIVIN, R., and ROTHMAN-DENES,L. B. (1978). Novel template requirements of N4 virion RNA polymerase. Proc. NatL Acad Sci USA 75, 3220-3224. GORDON,P., and RABINOWITZ,M. (1973). Evidence for deletion and changed sequence in the mitochondrial DNA of a spontaneously generated petite mutant of S mrevisiae. Biochemistry 12,116-D%. GOULIAN, M., LUCAS,Z., and KORNBERG,A. (1968). EnACKNOWLEDGMENT zymatic synthesis of deoxyribonucleic acid. XXV. This work was supported by USPHS Grants AI Purification and properties of deoxyribonucleic acid 12575, CA 19265, and GM 35170 to L.B.R.-D. polymerase induced by infection with phage T4. J. Bid C?wm 243,627-638. HAYNES,L. L., and ROTHMAN-DENES,L. B. (1985). N4 REFERENCES virion RNA polymerase sites of transcription initiation. Cell 41,597-605. ALBERTS,B., and FREY, L. (1970). T4 bacteriophage HILLEBRAND,G., MORELLI,G., LANKA, E., and SCHERgene 32: A structural protein in the recombination ZINGER,E. (1979). Bacteriophage T7 DNA primase: and replication of DNA. Nature (Landon) 227,1313A multifunctional enzyme involved in DNA repli1318. cation. Cold Spring Harbor Symp. Quant. BioL 43, ARAIU, H., and OGAWA,H. (1981).A l’7 Amber mutant 449-459. defective in DNA-binding proteins. MoL Gen Gend HUANG,C.-C., HEARST,J., and ALBERTS,B. (1981).Two 183,66-73. types of replication proteins increase the rate at BARRY, J., and ALBERTS,B. (1972). In vitro complewhich Td DNA polymerase traverses the helical rementation as an assay for new proteins required gions in a single-stranded DNA template. J. BioL for bacteriophage T4 DNA replication: Purification Chem. 256,4087-4094. of the complex specified by T4 genes 44 and 62. Proc JACOB,F., and BRENNER,S. (1963). Sur la regulation Nat1 Acad Sci USA 69,2717-2721. de la synthese du DNA chez les batteries: l’hyBOLLE,A., EPSTEIN,R., SALSER,W., and GEIDUSCHEK, pothese du replicon. C. R Acad. Sci 256,298-300. E. P. (1968). Transcription during T4 development: KLOBUTCHER,L. A., SWANTON,M. T., DONINI, P., and Synthesis and relative stability of early and late PRESCOTT,D. M. (1981). All gene-sized DNA moleRNA. J. Md Biol31,325-348. cules in four species of hypotrichs have the same BOTSTEIN,D. (1968). Synthesis and maturation of terminal sequence and an unusual 3’ terminus. Pm phage P22 DNA. I. Identification of intermediates. NatL Acad Sti USA 78.3015-3019. J. MoL Bid 34,621~641. CARL,P. L. (1970).Escherichia coli mutants with tem- KOLODNER,R., MASAMUNE,Y., LECLERC, J. E., and RICHARDSON,C. C. (1978). Gene 4 protein of bacteperature sensitive synthesis of DNA. Mol. Gen Geriophage T7: Purification, physical properties, and net. 109.107-116. stimulation of T7 DNA polymerase during the DENHARDT,D. T., DRESSLER,D. H., and HATHAWAY, elongation of polynucleotide chains. J. Bid Chem A. (1967). The abortive replication of &X174 DNA 253,566-573. in a recombination-deficient mutant of E. coli. Proc Nat1 Ad Sci USA 57,813-820. KONRAD,E. B., and LEHMAN,I. R. (1974).A conditional DERMODY,J. J., ROBINSON,G., and STERNGLANZ,R. lethal mutant of Esche-ri-schiacoli K12 defective in the 5’-3’ exonuclease associated with DNA poly(1979). Conditional lethal deoxyribonucleic acid merase I. Proc NatL Ad Sti USA 71,2048-2051. mutant of Escherichia wli. J BacterioL 139, 701704. KRELJZER,K., and HUANG, W. M. (1983). T4 topoisoDUNN,J. J., and STUDIER,F. W. (1981). Nucleotide semerase. In “Bacteriophage T4” (C. Mathews, E. quence of the genetic left end of bacteriophage T7 Kutter, G. Mosign, and P. Berget, eds.), pp. 90-96.
lished results). It is possible that these DNA fragments are the result of lagging strand synthesis, although a mechanism involving discontinuous replication of both DNA strands cannot be ruled out. Current efforts are directed at determining the functions of the N4 dn.s and N4 exe gene products, at examining in viva replicating N4 DNA molecules using electron microscopy and reconstituting N4 replication in vitro from purified proteins. These investigations should lead to an understanding of the mechanism of N4 DNA replication and the generation of its unusual 3’ termini.
44
GUINTA
American Society of Microbiology, Washington, D. C. KREUZER,K. N., MCENTEE,K., GEBALLE,A., and CozZARELLI,N. R. (1978). Lambda transducing phages for the n&l gene of E. coZiand conditional lethal nol.4 mutations. Md GermGonet. 167,129-137. LECHNER,R. L., and RICHARDSON,C. C, (1983). A preformed, topologically stable replication fork: Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein. .I. Biol Chew. 258,11185-11196. LIU, C. C., and ALBERTS,B. (1981). Characterization of RNA primer synthesis in the T4 bacteriophage in vitro replication system. J. Biol Chm 256,28212829.
ET AL. REUBEN,R. C., and GEFTER,M. L. (1974). A deoxyribonucleic acid-binding protein induced by bacteriophage T7: Purification and properties of the protein. J. Bid
Chem 249,3843-3850.
RICHARDSON, C. C. (1983). Bacteriophage T7: Minimal requirements for the replication of a duplex DNA molecule. Cell 33, 315-317. RIST, J. K., GUINTA,D. R., SUGINO,A., STAMROULY, J., FALCO, S. C., and ROTHMAN-DENES,L. B. (1983). Bacteriophage N4 DNA replication. In “Mechanisms of DNA Replication and Recombination” (N. R. Cozzarelli, ed.), pp. 245-254. Liss, New York. ROBERTS,R. J. (1985). Restriction and modification enzymes and their recognition sequences. Nucleic Ad Res. (Suppl. 13) r165-r200. ROMANO,L., and RICHARDSON,C. C. (1979). Requirements for the synthesis of ribonucleic acid primers during lagging strand synthesis by the DNA polymerase and Gene 4 protein of bacteriophage T7. J.
MACE,D. C., and ALBERTS,B. (1984). Characterization of the stimulatory effect of T4 gene 45 protein and the gene 44/62 protein complex on DNA synthesis by T4 DNA polymerase. J. Mol. Biol. 177,313-327. Biol. Chem 254,10476-10482. MARIANS,K. J. (1984).Enzymology of DNA replication in prokaryotes. CRC Crit Rev. Biochem. 17, 153- ROTHMAN-DENES, L. B., and SCHITO,G. C. (1974).Novel 215. transcribing activities in N4-infected E. CO&. Vi~olMASAMUNE,Y., FRENKEL, G. D., and RICHARDSON, OQY60,65-72. C. C. (1971). A mutant of bacteriophage T7 deficient SCHITO,G. C. (1973). The genetics and physiology of in polynucleotide ligase. J. BioL Chem. 246, 6874coliphage N4. virology 55,254-265. 6879. SILVER, L., and NOSSAL,N. (1978). DNA replication MATSON, S. W., TABOR, S., and RICHARDSON,C. C. by bacteriophage T4 proteins: Role of the DNA-de(1983). The gene 4 protein of bacteriophage T7, lay gene 61 in the chain initiation reaction. CoW, characterization of the helicase activity. J. Biol. Spring Harbor Symp. Quant. Biol. 43,489-494. Chem 258,14017-14024. SILVER,L., and NOSSAL,N. (1982). Purification of bacteriophage T4 gene 61 protein. A protein essential MODRICH,P., and RICHARDSON,C. C. (1975). Bacteriofor synthesis of RNA primers in the T4 in vitro phage T7 DNA replication in vitro: Bacteriophage replication system. J. Biol. Chem 257,11696-11705. T7 DNA polymerase, an enzyme composed of phageand host-specified subunits. J. Biol Chem 250,5515YENKATESAN,M., SILVER,L., and NOSSAL,N. (1982). 5522. Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a helicase. f. Bid MORRIS,C., MORAN,L., and ALBERTS,B. (1979). PuChem 257,12426-12434. rification of gene 41 protein of bacteriophage T4. J. Bid Chem 254,6797-6802. WECHSLER,J. A., and GROSS,J. (1971). Eschmischia coli temperature sensitive mutants for DNA synNOSSAL,N. (1979). DNA replication with bacteriothesis. Mel Gen Genet. 113,273-284. phage T4 proteins. Purification of the proteins encoded by T4 genes 41, 45, and 62 using a comple- ZEHRING,W. A., FALCO, S. C., MALONE, S. F., and ROTHYAN-DENES,L. B. (1983). Bacteriophage N4mentation assay. J. Biol Chem 254,6026-6031. induced transcribing activities in E co& III. A third OKAZAKI,T., KUROSAWA,Y., OGAWA,T., SEKI, T., SHIcistron required for N4 RNA polymerase II activity. NOZAKI,K., HIROSE,A. A., KOHARA, Y., MACHIDA, Virology 126,678-687. Y., TAMANOI,F., and HOZUMI,T. (1978). Structure and metabolism of the RNA primer in the discon- ZIVIN, R., MALONE, C., and ROTHMAN-DENES,L. B. (1980). Physical map of coliphage N4. firology 104. tinuous replication of prokaryotic DNA. Cold Spring 205-218. Harbor Symp. Quant. Biol43,205-219. ZIVIN, R., ZEHRING,W., and ROTHMAN-DENES,L. B. REED,R., and GRINDLEY,N. D. F. (1981). Transposon(1981). Transcriptional map of Bacteriophage N4: mediated site specific recombination in witro: DNA Location and Polarity of N4 RNAs. J. Mol. Bid 152. cleavage and protein-DNA linkage at the recom335-356. bination site. CeU25,721-728.
150,33-44 (1986)
Host and Phage-Coded Functions Required for Coliphage N4 DNA Replication D. GUINTA,’
J. STAMBOULY: S. C. FALCO: L. B. ROTHMAN-DENES4
J. K. RIST,
AND
Departmenta of Molecular Ba,
Genetics and Cell Biologzl and of Biochemistry and Molecular The University of Chicago, Chicago, Illinois 60637
Received October 10, 1985; acwpted Now&e-r
27, 1985
Escherichia coli strains containing mutations in various deoxyribonucleic acid synthesis cistrons have been tested for their ability to support bacteriophage N4 growth and, specifically, N4 DNA synthesis. N4 DNA synthesis is independent of the activity of the products of the E. coli dnaA, dnaB, dnaC, dnaE, dnaG, and rep genes. In contrast, N4 DNA replication requires the products of the dnaF, (ribonucleotide reductase) and lig (DNA ligase) genes of E. co& N4 DNA replication, specifically processing of short DNA fragments requires the 5-3 exonuclease activity of the po&f gene product. However, its DNA polymerizing activity is not required. In addition, the sensitivity of N4 DNA synthesis to inhibitors or temperature-sensitive mutants of E, coZi DNA gyrase suggests that this activity is required for N4 DNA synthesis. To date, we have found five N4 gene products required for N4 DNA replication: d&n (a single-stranded DNA binding protein), dnp (a DNA polymerase), dn.s (unknown function), vRNAp (the N4 virion-associated, DNA-dependent RNA polymerase) and ezro (a 5’-3’ exonuclease) 0 19% Academic PWS. Inc. INTRODUCTION
host DNA-dependent RNA polymerase (Rothman-Denes and Schito 1974; Zivin et a& 1981). Early RNAs appear in cells pretreated with chloramphenicol and rifampicin, indicating that neither an active E. coZi RNA polymerase nor translation of the phage genome is required for early RNA synthesis. The chloramphenicoland rifampicin-resistant transcription is due to a virion-encapsulated, DNA-dependent RNA polymerase (Falco et aL, 1977). In this paper we show that this RNA polymerase is directly required for N4 DNA replication as well. Moreover, in order to understand how N4 DNA synthesis is controlled, we have studied the host and phage requirements for N4 DNA synthesis.
N4 is a lytic bacteriophage that infects coli K 12 strains. Its virions contain 72 kb of linear double-stranded DNA with 400 to 450 bp of direct terminal repeats (Zivin et al, 1980). The structure of the left (or early) end of the genome is unique containing a 7-base 3’ overhang (L. Haynes, unpublished). The right end is variable; there are at least five different ends differing from each other by 10 bp giving rise to variability in the length of the terminal repeats (H. Ohmori and L. Haynes, unpublished). N4 differs from other DNA-containing bacteriophages in that synthesis of its early and middle RNAs does not require the activity of the Escherichia
MATERIALS
1 Present address: Department of Genetics, Stanford University School of Medicine, Stanford, Calif. ’ Present address: University of Pittsburgh, Pittsburgh, Pa. a Present address: E. I. duPont de Nemours Central Research, Wilmington, Del. ‘To whom requests for reprints should be addressed.
AND
METHODS
Baderial and phage strains. E. coli strain CR34 and its derivatives X9258 (dnaA46), JW184 (dnaB43), E486 (dmE486) were obtained from J. Wechsler (Wechsler and Gross, 1971). The rep 3 derivative of CR 34, NT rep 3, was provided by M. Gellert (Den33
004%6822/86 $3.00 Copyright All rights
8 19% hy Academic Press, Inc. of reproduction in any form reserved.
34
GUINTA
hardt et aL, 1967). M21 (DllO polA endol) and its dnuF El01 derivative, isolated by J. Wechsler (Wechsler and Gross, 1971) were obtained from R. Moses. DG 76 and its derivatives PC2 (dnaC2) and PC3 (dnuG3) were provided by P. Carl (Carl, 1970). H560 and its coumermycin-resistant derivative was constructed and provided by C. Peebles (Falco et a& 1978). KS463 and RS5064, its polAex1 derivative, were obtained from B. J. Bachmann, E. co,?iGenetic Stock Center, Yale University (Konrad and Lehman, 1974). GR523 and its Zigts251 derivative, GR501 were kindly provided by R. Sternglanz (Dermody et aL, 1979). N4 am12 was isolated by G. C. Schito. N4 33 am7, N4 am257, and N4 ts150 (Falco et al, 1977) were isolated by hydroxylamine mutagenesis in this laboratory. Media. M9 minimal salts medium (Bolle et aL, 1968), containing 0.4% glucose, 0.2% casaminoacids, 5 pg/ml thiamine, and 20 @g/ml thymine was used in all experiments. Chemicals. Coumermycin was a gift of Dr. N. C. Cozzarelli. [methyl-3H]Thymidine (50-62 Ci/mmol) was purchased from Schwarz-Mann. Lj3H]Aminoacid mix (1 mCi/ml) and En3Hance were purchased from New England Nuclear Company. Burst measurements. E. coli strains were grown at the permissive temperature in M9 minimal medium to a density of approximately 5 X lo* cells/ml (ODsm = 0.5). Cells were centrifuged and resuspended in fresh M9 medium prior to infection. The resuspended cells were divided in half, one-half of the cells were placed at 33” and the other half was placed at 43”. Parental strains and strains with immediate-stop dna ts mutations (dnuB, dnuE, dnuF) were preincubated at the restrictive temperature for 15 min, while the delayed-stop dnu strains were preincubated at the restrictive temperature for 60 min. After this period, wild type N4 phages were added to the cultures at a multiplicity of infection of 10. Unadsorbed phage were inactivated with N4 specific antiserum (final K = 10) added 5 min postinfection. The cells were incubated for an additional 5 min and then an aliquot of each infected culture was diluted 105fold into warmed M9 minimal media. At
ET AL.
this time, an aliquot was immediately plated E. coli W3350 to determine the number of infectious centers. The diluted cultures were incubated a total of 3 hr, after which samples were removed and the cells lysed with chloroform. Burst size was determined by titering the lysate on w3350. Measurement of the rate of DNA suntheti. Cells were grown and resuspended in M9 minimal media as described above. In the case of dnu ts mutants, thymine was added to a final concentration of 100 pg/ ml. After preincubating and treating the cells as above, N4+ was added at a multiplicity of 10. At various times after infection, 0.2-ml samples were removed and incubated for 2 min with 5 &i of [‘HIthymidine. At the end of the pulse, 0.05-ml samples were treated as described previously (Rothman-Denes and Schito, 1974). Alkaline sucrose gradients. Cells were infected and gradients run according to the method of Okazaki et al. with monitor modifications (Okazaki et uL, 1978). E. coli was grown to an ODszoof 0.5 in M9 media at 33” and infected with phage at an m.o.i. of 10. Thirty-six mintues after infection, the culture was shifted to 43” and pulsed 4 min later with 10 &i [3H]thymidine/ml of culture for lo- or 30-set intervals. In pulse and chase experiments, cells were pulsed for 10 set or 1 min, followed by a 4min chase with 2 mg/ml of unlabeled thymidine. Samples were removed and lysed with alkali according to Masamune et al. (1971). Cells (0.1 ml) were added to 0.1 ml ice cold 10 N NaOH, 0.5 1MEDTA, 0.1 M NaCN. After a lo-min incubation on ice, 0.1 ml 20% Sarkosyl NL97 was added along with [14CjN4DNA as a marker. Each sample was layered on a prechilled 9.0 ml linear 5-20s w/v sucrose gradient overlaying a 1.0 ml 80% w/v sucrose cushion. Sucrose solutions contained 0.1 M NaOH, 0.9 M NaCl, and 1 m&f EDTA. Gradients were centrifuged at 4” for 16 hr at 26,000 rpm in an SW41 rotor. Gradients were collected in 11 drop fractions from the bottom of the tube and acid-insoluble radioactivity was measured. Polyucrylumide gel electrophoresis and uutorudiogruphy. E. coli W3350 cells were
COLIPHAGE
N4 DNA
treated with rifampicin (200 rg/ml) 20 min before infection. Two minutes after infection with the indicated phages, L-[~H]aminoacid mix was added to 50 &i/ml. Infected cells were incubated for 60 min, collected by centrifugation, resuspended, and eleetrophoresed on a 15% polyacrylamide gel as described (Falco and RothmanDenes, 1979). The gel was prepared for fluorography by treatment with En’Hance according to manufacturer’s instructions. Extraction of labeled intracellular DNA and DNA-DNA h&ridimtim One-milliliter samples were treated according to Botstein (1968) except that after dialysis, RNAse was added to 0.1 mg/ml and incubated for 5 min at 37’ followed by 3 min at 70”. Sarkosyl was then added to 2% and the incubation was continued for 20 min at 70’ followed by 1 hr at 37”. Pronase was added to a final concentration of 2 mg/ml. The samples were incubated for 3 hr at 37” and dialyzed overnight against 10 m&Z Tris-HCl pH 8,10 mMEDTA, 0.15 MNaCI. DNA filters containing 10 pg of E. coli DNA or 20 gg of N4 DNA were prepared according to Gordon and Rabinowitz (1973). The labeled DNA was denatured with alkali. Hybridization was carried out in a total volume of 1 ml containing 1X SSC, 1% SDS at 65” for 14 hr. RESULTS
Production upon Ir@ectim of E. coli dnats Mutants under Restrictive cditim
N4 Progeny
To study the requirement of E. coli dna gene products for N4 growth, E. u& strains carrying temperature-sensitive mutations in various dna genes were tested at the restrictive temperature for their ability to support N4 growth. Table 1 shows the burst size of N4 upon infection of these strains as plaque-forming units per infective center. For dnaA, dnaB, and dnaG mutants, more progeny were made at 43O than at 33’. For the dnaE and dnaC mutants a large number of phage were released at 43”, although the burst size was smaller. The polh mutation, which affects the DNA polymerase activity of the protein, or rep,
35
REPLICATION TABLE
1
N4 BURST SIZE IN E. wli dm” STRAINS Burst/ infective center Relevant genotype
Strain
Ratio 43”/33”
33O
43”
377 614 583 752
1174 745 1409 289 1625
3.11 1.21 2.42 0.38 -
546
463
CR34
Wild type
X9258 JW184 E486 NT516
dwz.446 dnaB43 dnaE486 (polQ rep 3
DG76 PC2 PC3
Wild type
dnaCi? dmG3
34
22
178
261
0.85 0.65 1.47
M21 ME431
PdAl pdA1, d&El01
943 148
905 0
0.96 0.00
KS463 RS5064
Wild type pdAt?Xl
400
1800 30
0.07
GR523 GR501
ligts251
1333 1200
1842 38
1.38 0.03
Wild type
a Conditions
-
-
as under Materials
-
and Methods.
which affects the helicase II activity, did not affect N4 growth. In contrast, infection of the E. coli dnuF and lig mutants did not yield any progeny phage at 43”. It therefore appears that N4 phage development does not absolutely require the activity of the gene products of the host genes dnaA, dnuB, dnaC, dnaE, and dnaG, and the DNA polymerase activity of the polA gene product. The activities of the E. coli enzymes ribonucleotide reductase and DNA ligase are, however, required. N4 DNA Sgnthesis in E. coli Mutant Hosts To examine the effects of the above mutations directly on N4 DNA synthesis, infected cells were pulse-labeled with r3H]thymidine. Immediately after N4 infection of E. coli CR34, [‘Hlthymidine incorporation decreased due to cessation of host DNA synthesis (Fig. IA). This was followed by a burst of incorporation due to N4 DNA synthesis (Fig. 1A). Pretreatment of cells with chloramphenicol prevented
36
GUINTA
ET AL.
TIME
(min)
FIG. 1. Rate of [‘Hlthymidine incorporation after N4 infection of wild type and temperaturesensitive DNA synthesis mutants of E. co& at 43”. Conditions and strains as under Materials and Methods. (A) 0, wild type; 0, dnuAts; A, debts; n , dnuEts. (B) 0, wild type; 0, dnacts; A, dnaGts.
both the termination of host DNA synthesis as well as the initiation of N4 DNA synthesis, suggesting that both processes require phage protein synthesis (RothmanDenes and Schito, 1974). N4 DNA synthesis occurred normally when dnaA, drmB, and dnaE mutants were infected at 43” (Fig. lA), where the pertinent proteins were no longer functional in host DNA synthesis. Figure 1B shows the profile of rH]thymidine incorporation after N4 infection of the dnaC and dnaG temperature-sensitive derivatives of E. coli DG’76. Again, despite the absence of the respective functional dna gene products, N4 DNA contin-
TIME
FIG. 2. Rate of [8H]thymidine mutants E. coZi under restrictive (B) 0, kg+; 0, Zigts.
ues to be synthesized at the restrictive temperature. Similar results (not shown) are obtained upon infection of E. coli cells carrying rep mutations. Figure 2A shows the pattern of incorporation for mutants deficient in the polymerizing activity of DNA polymerase I and in ribonucleotide reductase. In strain M21, carrying the @Al mutation, significant levels of DNA synthesis occurred at both 33” (not shown) and 43”. However, the ME431 derivative of M21, which bears a mutation in dnaF, failed to support N4 DNA synthesis at 43”. Similar results were obtained when a strain of E. coli carrying
(min)
incorporation after N4 infection of &Al, dmF (A) and Zig (B) conditions. Infections were at 43“ (A) 0, @Al; 0, por!Al dn&ts.
COLIPHAGE
N4 DNA REPLICATION
37
a temperature-sensitive mutation in DNA ligase, &ts251, was infected at the nonpermissive temperature (Fig. 2B). These data therefore demonstrate that N4 DNA synthesis does not require the host dnaA, dnaB, dnaC, dnaE, dnaG, rep, and polA functions but requires functional ribonucleotide reductase and DNA ligase. Involvement of the 5’4 Ex~ucleolytic Activitg of DNA Polgwase I in N,$DNA &@thesis As shown above, the DNA polymerizing activity of polA is not required for N4 growth. However, no N4 was produced upon infection of E. coli cells deficient in the 5’3’ exonuclease activity of mL4 (Table 1). This result was observed in polAexl cells, which have a temperature-sensitive, conditional lethal mutation in the DNA polymerase I 5’-3’ exonuclease, as well as in polA12 cells which are defective in coordinating the polymerase and 5’-3’ exonuclease activities of Pol I. The pattern of r3H]thymidine incorporation after N4 infection of these mutant strains, however, was norma (not shown). In order to explain this discrepancy, we analyzed the accumulation of N4 Okazaki fragments in wild type or exonuclease mu-
IO
20
10
30
20
JO
i=RAcTulN
FIG. 3. Effect of E. coZipdrlez1 mutation on N4 DNA synthesis. (A) E. wli KS463, (B) E. co& RS5064, a polAex1 derivative of KS463. Cells were infected at 43” with wild type N4 and intracellular DNA was labeled for 10 set (0) or 30 see (0). Cells were lysed and alkaline sucrose gradients were run as described under Materials and Methods. Virion length N4 DNA was found in fractions 1-3 in all gradients. Sedimentation is from right to left.
FIG. 4. Pulse and chase of labeled N4 DNA in polAex1 cells. E. coli RS5064 was infected with wild type N4 at 43”. Cells were labeled with [BHjthymidine for 10 set (A) or 1 min (O), followed by a I-min chase with an excess of unlabeled thymidine (0). Cells were lysed and alkaline sucrose gradients were run as under Materials and Methods. Virion length DNA was found in fractions 1-3. Sedimentation was from right to left.
tant infected cells, since the 5’-3’ exonuclease activity of polA is involved in that process in other phage systems. The results of this experiment are shown in Fig. 3. Small DNA fragments did not accumulate in the wild type parental strain. However, they did accumulate in both the polAex1 and polAl2 (not shown) mutants under restrictive conditions. Figure 4 shows that these small fragments accumulating in polAex1 cells infected with N4 could be chased into larger DNA, demonstrating the slow conversion of nascent DNA into mature DNA rather than a slow process of degradation. This suggests that these small fragments are indeed Okazaki fragments. The 5’-3 exonuclease of the host DNA polymerase I is, therefore, required to process N4 Okazaki fragments. Efect of DNA Gyrase Inhibitors DNA Synthesis
on N4
N4 early RNA synthesis, which is required for all subsequent events during N4 infection, requires active E. coli DNA gyrase (Falco et al, 1978). In order to assess the possible involvement of E. co2i DNA gyrase in N4 DNA synthesis, gyrase inhibitors were added after the onset of DNA replication (25 min postinfection). Coumermycin is a strong inhibitor of N4
33
GUINTA
ET AL.
DNA synthesis. Maximal inhibition was achieved within 5 min of the addition of the drug (Fig. 5). The effect was not observed in coumermycin-resistant infected cells, suggesting that the effect is through inhibition of E. coli DNA gyrase, and that its activity is required for N4 DNA replication. Similar results were obtained after shift-up to the restrictive temperature of infected cells carrying temperature sensitive mutations in the ggrA locus (not shown). Involvement of N&%ded DNA Replication
Functions
in TIME
(min)
FIG. 6. Rate of mjthymidine incorporation after At present, suppressor-sensitive mutants of N4 have been isolated in eight cis- N4 mutant infection of E. cd W3350. (A) 0, N4+; n , trons which are required for DNA repli- um15; A, am.23 0, am126. (B) 0, awt257; A, am33A7; cation. Mutations in one cistron, coding for l , am12. Other conditions as under Materials and Methods. a 5’-3’ exonuclease, show a temperaturedependent DNA arrest phenotype and will not be discussed further here (D. Guinta, alleles am126, am15, and am23, are reS. Spellman, and L. Rothman-Denes, un- quired for N4 middle RNA synthesis published). Mutations in the seven other (Rothman-Denes and Schito, 1974; Zivin et cistrons express a DNA negative pheno- c& 1981; Zehring et al, 1983). Results of type, i.e., no N4 DNA synthesis is detected experiments with temperature-sensitive (Fig. 6A). Three of these cistrons, rnaII a, mutants in these three latter cistrons inrnaII b, and rn.uII c, represented by mutant dicate that their gene products are not directly involved in N4 DNA replication since inactivation of the product, after middle RNA synthesis has occurred, has no effect on N4 DNA replication (not shown). The products of three other N4 cistrons, dnp (a DNA polymerase), &n (DNA binding protein), and dns (unknown function), represented by mutants am257, am33A7, and am12, are directly involved in DNA replication (Fig. 6B) (Schito, 1973). The products of two of these genes can be easily identified on polyacrylamide gels when the pattern of proteins labeled after infection of suppressor and nonsuppressor containing strains are compared (Fig. 7). The product of the dns cistron is probably a 1 1 protein of 78,000 mol wt (~2) since it is 40 so 10 20 JO missing in am12 infected cells. An abunTIME (min) dant protein, (~8: 30,000 mol wt) is missing FIG. 5. Effect of coumermycin on N4 DNA synthesis. after awz33A7infection, suggesting that it Two-minute pulses of @IJthymidine were performed is the product of the dnp cistron. No difas described under Materials and Methods. Coumerferences could be detected after am257 inmycin (20 g/ml) was added at 25 min after infection of E. coZiH566 mall (A) or E. cm.5 H566 m&l CaL’ (B). fection. This is not due to the particular n , no coumermycin; 0, coumermycin. mutant infection used since similar results
COLIPHAGE
N4 DNA REPLICATION
39
polymerase is resistant (Falco et aL, 1977). Further characterization of N4ts150 revealed that this enzyme pIays a second role in N4 development. The effect of raising the temperature on the rate of DNA synthesis after N4 wild type and N4ts150 infection is compared in Fig. 8. When E. coli cells were infected with wild type N4 at 33”, pulse-labeling with [SH)thymidine yielded a characteristic pattern shown in Fig. 8A. A similar pattern was obtained upon infection at 43”. When cells infected at 33” were shifted to 43” at 25 min after t ~8 infection, no change in the pattern of thymidine incorporation was observed. Infection with N4ts150 yielded strikingly different results (Fig. 8B). At the permissive temperature the pattern was similar to that observed after N4 wild type infection. In contrast, at 43”, a slow decline in the rate of thymidine incorporation was seen. The rapid increase in rate, characteristic of the onset of viral DNA replication, did not occur. This result was expected because the virion RNA polymerase, which am u N4 is inactive at 43“, is required for N4 early 257 T2” &ii RNA synthesis and therefore for all postFIG. 7. N4 induced proteins after wild type and mu- infection protein synthesis. When cells intant infection. Conditions as under Materials and fected at 33” were shifted to 43” at 25 min Methods. U: uninfected control. PO: indicates place of after infection, the rate of thymidine inmigration of a protein which has been identified as corporation rapidly decreased to less than the dnp gene product (K. Rist, unpublished). P2: pro10% of the level attained at 33”. tein missing in dns mutant (am12) infection. P8: proThe effect of temperature shift on DNA tein missing in &p mutant (aWA7) infection). synthesis in N4ts150 infected cells was not indirectly due to the inhibition of synthesis were obtained with other dnp mutants, of a protein required for DNA replication suggesting that the product of dnp is either or maturation. Addition of chloramphentoo scarce to be detected by this technique icol at 25 min after infection to a wild type or it comigrates with other phage or cel- N4 or N4ts150 infected culture did not inhibit N4 DNA synthesis, although an inlular proteins. hibition of the increase in the rate of DNA replication was observed. Requirement of the N4 virion-encapsulated The cessation of rH]thymidine incorRNA polgmerase in N4 DNA replicaporation, which followed a shift to the retion strictive temperature in N4ts150 infected N4ts150 is a temperature-sensitive mu- cells, was specific for N4 DNA synthesis. tant of N4 which shows temperature sen- Table 2 shows the results of hybridization sitive early N4 RNA synthesis (Falco et al, of DNA extracted from wild type N4 or 1977). We have shown that this mutation N4ts150 cells to either E. co&ior N4 DNA. resides in the N4 virion-encapsulated RNA DNA labeled from 15 to 20 min, or 35 to 40 polymerase since the enzyme purified from min after N4 or N4ts150 infection at 36”, N4ts150 virions is sensitive to thermal in- hybridized exclusively to N4 DNA. When activation while wild type N4 virion RNA the temperature was shifted from 36 to 43”
40
GUINTA
ET AL.
TIME AFTER INFECTION
bth)
FIG. 8. Rate of [%I]thymidine incorporation in wild type or ts150 N4 infected cells. l , infection at 33’; 0, infection at 43’; A, infection at 33’, chloramphenicol was added at the time indicated by the arrow; (x, infection at 33”, temperature shifted to 43” at the time indicated by the arrow. Other conditions as described under Materials and Methods.
at 21 min after wild type N4 infection and the DNA was labeled from 35 to 40 min, the DNA that was synthesized hybridized to only N4 DNA. In contrast, when the temperature was shifted during N4ts150 infection, very little label was incorporated into DNA during the pulse and none of it hybridized to N4 DNA. Genetic evidence indicates that the double phenotype exhibited by N4ts150-i.e., temperature-sensitive early RNA synthesis and DNA replication-is due to a single mutation. It has not been possible to separate the two phenotypes by back-crossing N4ts150 to wild type N4. Reversion of both phenotypes occurs simultaneously at a frequency of 3 X 10e5.Furthermore, a second independently isolated N4 mutant, N4ts245, has the same double phenotype. Reversion of both phenotypes in N4ts245 occurs simultaneously at a frequency of 2 X 10e4 (data not shown). Genetic recombination indicates that N4ts150 and N4ts2.45are two different but closely linked mutations (data not shown). We therefore conclude that the N4 virion RNA polymerase plays a direct role in N4 DNA replication and mutations in it define a new cistron with a DNA negative phenotype.
DISCUSSION
The large N4 genome is unusual because of the structure of its termini. The virion DNA is ‘72kb in length and contains direct terminal repeats of 400-450 bp (Zivin et aL, 1980). The genetic left end of the genome (early region) is unique and contains a ‘7 base single-stranded 3’ end (3’CCATAAA) (Ohmori and Haynes, unpublished). The TABLE 2 ANALYSIS OF HYBRIDIZATION
DNA
SYNTHESIS BY DNA-DNA IN N4 INFECCED E. wli CELLS cpm hybridized to
Temperature 36’ 36’
43Ob
Labeling period (min)
Phage
Input cpm
-5-o 15-20 15-20 35-40 35-40 35-40 35-40
N4+ ts150 N4+ te.150 N4+ ts150
1341 1582 1414 1704 1394 202.3 147
N4 DNA’ 5 1073 666 1556 912 1252 0
E. wli DNA” 2.22 15 13 4 18 30 34
a Blanks, 24-56 cpm, were subtracted. ’ 36’C infected cultures were shifted to 43’ at 21 min after infection. Other conditions as under Materials and Methods.
COLIPHAGE
N4 DNA
right end is not unique; one major and four minor ends exist in the population, which differ by 10 bp over a 50-nucleotide-long region. These ends also have 3’ singlestranded overhangs (Ohmori and Haynes, unpublished). Although 3’ tails are common as a result of restriction endonuclease (Roberts, 1985) or topoisomerase cleavage (Reed and Grindley, 1981), they are not found at the ends of other phage and viral genomes. N4 shares its unusual terminal structure with the macronuclear DNAs of hypotrichous ciliates, such as Oxgtricha nova which has 16 base 3’ single-stranded tails of the sequence G4T4G4T4(Klobutcher et al, 1981). In order to understand how N4 DNA replicates and generates its 3’ singlestranded ends, we have determined those E. coli functions which are required for N4 DNA replication as well as whether N4 codes for any replication functions of its own. By analysis of phage burst size and measurement of N4 DNA synthesis we have shown that N4 does not require the activity of the host dnuA, dnaB, dnaC, dnaE, dnuG, rep, or polA gene products. The lack of a requirement for either of the host DNA polymerases III or I (dnaE or polA) strongly suggests that N4 codes for its own DNA polymerase. The independence from dnaG, dnuB and dnuC products implies that N4 provides its own priming machinery as well. Finally, it is not surprising that the dnaA product is not required for N4 replication since specific replicons should be activated by specific initiation proteins (Jacob and Brenner, 1963). With the use of conditional-lethal mutants, five N4 genes have been shown to be required for N4 DNA synthesis thus far. Mutants in three genes, dns, dnp, and dbp, show a DO phenotype; that is, no N4 DNA synthesis is detectable. The function of the dnp and @genes has been determined using an in vitro N4 DNA replication system. While extracts made from wild-type N4 infected E. coli support specific replication of N4 DNA, extracts made from N4 mutants in the dnp or dbp genes do not. This has allowed the purification of the products of the dnp and dbp genes from wild type
REPLICATION
41
N4 extracts by complementation of the appropriate mutant extract (Rist et al, 1983). The purified proteins have been shown to be a DNA polymerase (drip) and DNA binding protein (d&n) (Rist et d, submitted). The product of the dns gene, although identified on SDS-polyacrylamide gels (Fig. 7), has not been purified, and its function is not known. Two other N4-coded functions required for N4 replication have been identified but the elucidation of their roles awaits an understanding of the mechanism of N4 DNA replication. The N4 exo gene codes for a 5’3’ exonuclease which can be purified by in vitro complementation for N4 DNA replication of an exo mutant-infected cell extract. Only one mutant allele of this gene has been isolated and it has a temperaturedependent DNA arrest phenotype (D. Guinta, S. Spellman, and L. B. RothmanDenes, unpublished). Analysis of the role of the N4 virion RNA polymerase in replication is complicated by its involvement in all early phage events. When this aspect of the virion polymerase’s function is separated from its effect on DNA synthesis by temperature shift-up experiments, it is clear that the polymerase is directly required for N4 replication. The virion RNA polymerase has been shown to accurately initiate transcription on a denatured N4 DNA template in vitro (Falco et aL, 1978; Haynes and Rothman-Denes, 1985). This enzyme will also accurately transcribe double-stranded DNA in vitro if the DNA carries a promoter and is supercoiled and E. cola’single-stranded DNA binding protein (ssb) is present (P. Markiewicz, J. Chase, unpublished). In tivo, transcription by this enzyme requires the activity of E. co& DNA gyrase (Falco et aZ., 1978), also suggesting a requirement for unwinding of the helix in the order for the polymerase to recognize its promoters. It is not yet known what replicative form of N4 DNA is the substrate for the virion polymerase in N4 replication, nor is it known whether this RNA polymerase is required for priming at the N4 origin of replication or for some step in possible lagging strand synthesis.
42
GUINTA
In general, when a virulent bacteriophage supplies its own DNA polymerase, it also supplies many of the proteins required to maintain a moving replication fork. The T7 replication fork uses only three phage-encoded proteins to maintain leading-strand synthesis and prime and maintain lagging-strand synthesis (Richardson, 1983). The priming (Hillebrand et al, 1979; Roman0 and Richardson, 1979) and helicase (Lechner and Richardson, 1983; Matson et aL, 1983) activities reside in a single, multifunctional protein, the gene 4 product (Kolodner et ah, 1978). The single-stranded DNA binding protein (Reuben and Gefter, 1974) is the product of gene 2.5 (Dunn and Studier, 1981). Its activity is dispensible since it can be substituted by the host DNA binding protein (Araki and Ogawa, 1981). The DNA polymerase activity resides in a complex composed of a phage-coded polypeptide, the gene 5 product, and E. coli thioredoxin (Modrich and Richardson, 1975). In contrast, the bacteriophage T4 replication fork requires the activity of seven phage-coded proteins. The T4 DNA polymerase (Goulian et ah, 1968) and singlestranded DNA binding (Alberts and Frey, 1970) proteins are both single polypeptides. However, three “accessory” proteins, the products of genes 45,44, and 62 (Barry and Alberts, 1972; Nossal, 1979), are required by T4 DNA polymerase for efficient polymerization. These proteins act as a “clamp” for T4 DNA polymerase, increasing the rate of polymerization and eliminating pausing in an ATP-dependent fashion (Alberts et ak, 1980; Huang et aL, 1981; Mace and Alberts, 1984). The T4 primase activity requires the combined action of the T4 genes 41 and 61 protein products (Morris et aL, 1979; Silver and Nossal, 1978, 1982; Liu and Alberts, 1981). Helicase activity is present in the 41 protein (Venkatesan et al, 1982). The N4 genome is large enough to code for most of its replication functions. We have identified two proteins required for maintenance of an active replication fork, a DNA polymerase and a single-stranded DNA binding protein. N4 probably encodes
ET AL.
its own primase and helicase as well. It will be interesting to see if these activities reside in replication genes that we have already identified or in, as yet, unidentified genes. Furthermore, as we have no mutant alleles in N4 recombination genes, we cannot assess the contribution, if any, of recombination to N4 DNA replication. Four host gene products are required for N4 DNA synthesis: DNA gyrase, ribonucleotide reductase, DNA ligase, and the 5’3’ exonuclease function of DNA polymerase I. The requirement of the host DNA gyrase for N4 replication was shown by the addition of the specific DNA gyrase inhibitors after the onset of N4 DNA replication. Because the N4 virion RNA polymerase requires the activity of DNA gyrase for transcription of the early region of the N4 genome in viva (Falco et cd, 1978), it is possible that DNA gyrase is also required to aid the virion polymerase in its replication function as well. However, DNA gyrase may be required directly for N4 DNA synthesis. T4 genes 39, 52, and 60, which encode proteins that form a type II topoisomerase, have mutant phenotypes characterized by a reduction in the rate of the initial phase of T4 DNA replication (reviewed in Kreuzer and Huang, 1983; Marians, 1984). T4 topoisomerase mutants are predominantly defective in initiation of replication through recombinational intermediates (G. Mosig, personal communication). The N4 requirement for ribonucleotide reductase probably serves to provide a pool of nucleotides for N4 DNA synthesis. N4 shuts off host DNA synthesis, but does not degrade the host genome (Rothman-Denes and Schito, 1974; see also Fig. 1). The requirements for the host ligase and DNA polymerase I 5’-3’ exonuclease, in conjunction with the demonstration of short nascent DNA in N4 infected cells, suggest that the mechanism of N4 replication includes some form of discontinuous DNA synthesis. These nascent DNA chains have a size similar to that of T7 Okazaki fragments (Okazaki et al, 1978) (1-6 kb) and can be chased into longer DNA molecules (M. S. Pearle and D. Guinta, unpub-
COLIPHAGE
N4 DNA REPLICATION
43
DNA to the beginning of gene 4. J. MoL BioL 148, 303-330. FAL.CO,S. C., and ROTHMAN-DENES,L. B. (1979). Bacteriophage Nd-induced transcribing activities in Eschmichia c&i I. Detection and characterization in cell extracts. Vi* 95.454-465. FALCO,S. C., VANDERLAAN, K., and ROTHYAN-DENES, L. B. (1977). Virion-associated RNA polymerase required for N4 development. Proc NatL AuuL Sci USA 74.520-52.X FALCO, S. C., ZIVIN, R., and ROTHMAN-DENES,L. B. (1978). Novel template requirements of N4 virion RNA polymerase. Proc. NatL Acad Sci USA 75, 3220-3224. GORDON,P., and RABINOWITZ,M. (1973). Evidence for deletion and changed sequence in the mitochondrial DNA of a spontaneously generated petite mutant of S mrevisiae. Biochemistry 12,116-D%. GOULIAN, M., LUCAS,Z., and KORNBERG,A. (1968). EnACKNOWLEDGMENT zymatic synthesis of deoxyribonucleic acid. XXV. This work was supported by USPHS Grants AI Purification and properties of deoxyribonucleic acid 12575, CA 19265, and GM 35170 to L.B.R.-D. polymerase induced by infection with phage T4. J. Bid C?wm 243,627-638. HAYNES,L. L., and ROTHMAN-DENES,L. B. (1985). N4 REFERENCES virion RNA polymerase sites of transcription initiation. Cell 41,597-605. ALBERTS,B., and FREY, L. (1970). T4 bacteriophage HILLEBRAND,G., MORELLI,G., LANKA, E., and SCHERgene 32: A structural protein in the recombination ZINGER,E. (1979). Bacteriophage T7 DNA primase: and replication of DNA. Nature (Landon) 227,1313A multifunctional enzyme involved in DNA repli1318. cation. Cold Spring Harbor Symp. Quant. BioL 43, ARAIU, H., and OGAWA,H. (1981).A l’7 Amber mutant 449-459. defective in DNA-binding proteins. MoL Gen Gend HUANG,C.-C., HEARST,J., and ALBERTS,B. (1981).Two 183,66-73. types of replication proteins increase the rate at BARRY, J., and ALBERTS,B. (1972). In vitro complewhich Td DNA polymerase traverses the helical rementation as an assay for new proteins required gions in a single-stranded DNA template. J. BioL for bacteriophage T4 DNA replication: Purification Chem. 256,4087-4094. of the complex specified by T4 genes 44 and 62. Proc JACOB,F., and BRENNER,S. (1963). Sur la regulation Nat1 Acad Sci USA 69,2717-2721. de la synthese du DNA chez les batteries: l’hyBOLLE,A., EPSTEIN,R., SALSER,W., and GEIDUSCHEK, pothese du replicon. C. R Acad. Sci 256,298-300. E. P. (1968). Transcription during T4 development: KLOBUTCHER,L. A., SWANTON,M. T., DONINI, P., and Synthesis and relative stability of early and late PRESCOTT,D. M. (1981). All gene-sized DNA moleRNA. J. Md Biol31,325-348. cules in four species of hypotrichs have the same BOTSTEIN,D. (1968). Synthesis and maturation of terminal sequence and an unusual 3’ terminus. Pm phage P22 DNA. I. Identification of intermediates. NatL Acad Sti USA 78.3015-3019. J. MoL Bid 34,621~641. CARL,P. L. (1970).Escherichia coli mutants with tem- KOLODNER,R., MASAMUNE,Y., LECLERC, J. E., and RICHARDSON,C. C. (1978). Gene 4 protein of bacteperature sensitive synthesis of DNA. Mol. Gen Geriophage T7: Purification, physical properties, and net. 109.107-116. stimulation of T7 DNA polymerase during the DENHARDT,D. T., DRESSLER,D. H., and HATHAWAY, elongation of polynucleotide chains. J. Bid Chem A. (1967). The abortive replication of &X174 DNA 253,566-573. in a recombination-deficient mutant of E. coli. Proc Nat1 Ad Sci USA 57,813-820. KONRAD,E. B., and LEHMAN,I. R. (1974).A conditional DERMODY,J. J., ROBINSON,G., and STERNGLANZ,R. lethal mutant of Esche-ri-schiacoli K12 defective in the 5’-3’ exonuclease associated with DNA poly(1979). Conditional lethal deoxyribonucleic acid merase I. Proc NatL Ad Sti USA 71,2048-2051. mutant of Escherichia wli. J BacterioL 139, 701704. KRELJZER,K., and HUANG, W. M. (1983). T4 topoisoDUNN,J. J., and STUDIER,F. W. (1981). Nucleotide semerase. In “Bacteriophage T4” (C. Mathews, E. quence of the genetic left end of bacteriophage T7 Kutter, G. Mosign, and P. Berget, eds.), pp. 90-96.
lished results). It is possible that these DNA fragments are the result of lagging strand synthesis, although a mechanism involving discontinuous replication of both DNA strands cannot be ruled out. Current efforts are directed at determining the functions of the N4 dn.s and N4 exe gene products, at examining in viva replicating N4 DNA molecules using electron microscopy and reconstituting N4 replication in vitro from purified proteins. These investigations should lead to an understanding of the mechanism of N4 DNA replication and the generation of its unusual 3’ termini.
44
GUINTA
American Society of Microbiology, Washington, D. C. KREUZER,K. N., MCENTEE,K., GEBALLE,A., and CozZARELLI,N. R. (1978). Lambda transducing phages for the n&l gene of E. coZiand conditional lethal nol.4 mutations. Md GermGonet. 167,129-137. LECHNER,R. L., and RICHARDSON,C. C, (1983). A preformed, topologically stable replication fork: Characterization of leading strand DNA synthesis catalyzed by T7 DNA polymerase and T7 gene 4 protein. .I. Biol Chew. 258,11185-11196. LIU, C. C., and ALBERTS,B. (1981). Characterization of RNA primer synthesis in the T4 bacteriophage in vitro replication system. J. Biol Chm 256,28212829.
ET AL. REUBEN,R. C., and GEFTER,M. L. (1974). A deoxyribonucleic acid-binding protein induced by bacteriophage T7: Purification and properties of the protein. J. Bid
Chem 249,3843-3850.
RICHARDSON, C. C. (1983). Bacteriophage T7: Minimal requirements for the replication of a duplex DNA molecule. Cell 33, 315-317. RIST, J. K., GUINTA,D. R., SUGINO,A., STAMROULY, J., FALCO, S. C., and ROTHMAN-DENES,L. B. (1983). Bacteriophage N4 DNA replication. In “Mechanisms of DNA Replication and Recombination” (N. R. Cozzarelli, ed.), pp. 245-254. Liss, New York. ROBERTS,R. J. (1985). Restriction and modification enzymes and their recognition sequences. Nucleic Ad Res. (Suppl. 13) r165-r200. ROMANO,L., and RICHARDSON,C. C. (1979). Requirements for the synthesis of ribonucleic acid primers during lagging strand synthesis by the DNA polymerase and Gene 4 protein of bacteriophage T7. J.
MACE,D. C., and ALBERTS,B. (1984). Characterization of the stimulatory effect of T4 gene 45 protein and the gene 44/62 protein complex on DNA synthesis by T4 DNA polymerase. J. Mol. Biol. 177,313-327. Biol. Chem 254,10476-10482. MARIANS,K. J. (1984).Enzymology of DNA replication in prokaryotes. CRC Crit Rev. Biochem. 17, 153- ROTHMAN-DENES, L. B., and SCHITO,G. C. (1974).Novel 215. transcribing activities in N4-infected E. CO&. Vi~olMASAMUNE,Y., FRENKEL, G. D., and RICHARDSON, OQY60,65-72. C. C. (1971). A mutant of bacteriophage T7 deficient SCHITO,G. C. (1973). The genetics and physiology of in polynucleotide ligase. J. BioL Chem. 246, 6874coliphage N4. virology 55,254-265. 6879. SILVER, L., and NOSSAL,N. (1978). DNA replication MATSON, S. W., TABOR, S., and RICHARDSON,C. C. by bacteriophage T4 proteins: Role of the DNA-de(1983). The gene 4 protein of bacteriophage T7, lay gene 61 in the chain initiation reaction. CoW, characterization of the helicase activity. J. Biol. Spring Harbor Symp. Quant. Biol. 43,489-494. Chem 258,14017-14024. SILVER,L., and NOSSAL,N. (1982). Purification of bacteriophage T4 gene 61 protein. A protein essential MODRICH,P., and RICHARDSON,C. C. (1975). Bacteriofor synthesis of RNA primers in the T4 in vitro phage T7 DNA replication in vitro: Bacteriophage replication system. J. Biol. Chem 257,11696-11705. T7 DNA polymerase, an enzyme composed of phageand host-specified subunits. J. Biol Chem 250,5515YENKATESAN,M., SILVER,L., and NOSSAL,N. (1982). 5522. Bacteriophage T4 gene 41 protein, required for the synthesis of RNA primers, is also a helicase. f. Bid MORRIS,C., MORAN,L., and ALBERTS,B. (1979). PuChem 257,12426-12434. rification of gene 41 protein of bacteriophage T4. J. Bid Chem 254,6797-6802. WECHSLER,J. A., and GROSS,J. (1971). Eschmischia coli temperature sensitive mutants for DNA synNOSSAL,N. (1979). DNA replication with bacteriothesis. Mel Gen Genet. 113,273-284. phage T4 proteins. Purification of the proteins encoded by T4 genes 41, 45, and 62 using a comple- ZEHRING,W. A., FALCO, S. C., MALONE, S. F., and ROTHYAN-DENES,L. B. (1983). Bacteriophage N4mentation assay. J. Biol Chem 254,6026-6031. induced transcribing activities in E co& III. A third OKAZAKI,T., KUROSAWA,Y., OGAWA,T., SEKI, T., SHIcistron required for N4 RNA polymerase II activity. NOZAKI,K., HIROSE,A. A., KOHARA, Y., MACHIDA, Virology 126,678-687. Y., TAMANOI,F., and HOZUMI,T. (1978). Structure and metabolism of the RNA primer in the discon- ZIVIN, R., MALONE, C., and ROTHMAN-DENES,L. B. (1980). Physical map of coliphage N4. firology 104. tinuous replication of prokaryotic DNA. Cold Spring 205-218. Harbor Symp. Quant. Biol43,205-219. ZIVIN, R., ZEHRING,W., and ROTHMAN-DENES,L. B. REED,R., and GRINDLEY,N. D. F. (1981). Transposon(1981). Transcriptional map of Bacteriophage N4: mediated site specific recombination in witro: DNA Location and Polarity of N4 RNAs. J. Mol. Bid 152. cleavage and protein-DNA linkage at the recom335-356. bination site. CeU25,721-728.