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Escherichia coli dnaG gene product is required for a normal rate of phage T4 DNA synthesis
VIROLOGY
123,443-447 (1982)
SHORT COMMUNICATIONS Escherichia
co/i dnaG Gene Product Is Required for a Normal Rate of Phage T4 DNA Synthesis CLAVE LILIEN’
Department
of Molecular and Medical Microbiology, Tucson, Arizona
University 85724
of Arizona College of Medicine,
Received May 25, 1982; accepted August 23, 1982 An Escherichia coli dnaG temperature-sensitive (ts) mutant, which produces a defective RNA primase, does not permit normal bacteriophage T4 growth and a normal rate of DNA synthesis. By contrast an E. coli dnaC ts mutant, a revertant of the dnoG ts mutant, and wild type allow a normal rate of phage T4 DNA synthesis. Evidence is presented that the phage gene 61 RNA primase and the host dnuG primase act independently of each other. We speculate that the host dnaG primase may be utilized for initiation of phage T4 DNA synthesis at replicative origins.
differences suggest that the two prim&es do not perform identical functions. There is evidence that some E. coli host replicative proteins are utilized in phage T4 DNA synthesis (6, 7). Breschkin and Mosig (6) showed that the E. coli dnaG primase is required for the residual primary DNA replication that occurs in a phage T4 gene 32 mutant infection. McCarthy (7), in our laboratory, showed that another E. coli replicative protein, the DNA gyrase, could partially substitute for the products of phage T4 genes 39,52, and 60, which jointly specify a topoisomerase (8). The phage topoisomerase, or substituting host gyrase, appear to be required for a step in initiation of phage DNA synthesis (7,9). The present experiments were undertaken to test whether the E. coli dnaG gene function is required in vivo for phage T4 DNA synthesis. The E. coli strains used in these experiments were PC2 (dnaC ts mutant), PC3 (dnaG ts mutant), PC3R (revertant of dnaG ts mutant), DG76 (wild type), and CR63 (wild type). These are listed in Table 1. PC2 (dnaC ts mutant) is defective in the initiation of replication in E. coli (1 l), and there is no a priori reason to expect it to affect phage T4 DNA replication. With the exception of CR63, these strains were obtained from the E. coli Stock Center, Yale
The product of the E. coli dnaG gene is an RNA primase (1, 2). When singlestranded circular DNA from phage G4 is used in vitro as a template, the dnaG primase synthesizes a 26 to 29 residue RNA transcript at the origin of replication (3). If primase action is coupled directly to replication, the RNA transcript is abbreviated to a few nucleotides by an early extension by DNA polymerase (2). Alberts et al. (4) have presented evidence that the product of phage T4 gene 61 is also an RNA primase that operates together with gene 41 protein, a DNA helicase, to synthesize RNA primers. These primers are pentaribonucleotides of general sequence pppApCpNpNpNp, which, in the presence of the remaining five replicative proteins, are utilized to prime de nouo T4 DNA chain starts during lagging strand synthesis (5). Although the E. coli dnuG gene product and the product of phage T4 gene 61 both have primase activity, they also have several important differences (2). The phage T4 gene 61 primase has single-stranded DNA-dependent ATPase activity, and can utilize only rNTPS. The E. coli dnaG primase does not have such an ATPase activity, and can utilize dNTPs in the presence of rATP, as well as rNTPS. These 1 To whom reprint
requests should be addressed. 443
0042-6822/82/160443-05$02.00/O Copyright All rights
Q 1982 by Academic of reproductionin
Press, Inc. reserved.
any form
444
SHORT
COMMUNICATIONS TABLE
1
BACTERIAL STRAINS Strain
Relevant
genotype
Comments
Source
Reference
DG76
leuB6, thyA47, deoC3, rpsL153, X- sexF-
This strain is the parental type of PC2, PC3, and PC5
E. coli Stock Center
(10)
PC2
lcuB6, thyA47, dnaC2, deoC3, rpsL153, strA157, h- sexF-
dnoC2 is a temperaturesensitive mutant
E. coli Stock Center
(11)
PC3
leuB6, thyA47, dnuG3, deoC3, rpsL153, XserF-
dnaG3 is a temperaturesensitive mutant
E. coli Stock Center
(11)
PCBR
leuB6, thyA47, dnaG3 (rev), deoC3, rpsL153, A- sexF-
A temperatureinsensitive revertant of PC3
Obtained as described in text
CR63
supD60
Standard permissive host for phage T4 am mutants
This laboratory
University, New Haven, Connecticut, through the courtesy of Dr. Barbara Bachmann. The first evidence that E. coli dnaG gene product functions in phage T4 replication was seen in burst size experiments. E. coli cells were grown in Hershey broth (13) supplemented with 20 g/ml thymine (Hershey-supp), to about 2 X 107/ml at 30”, centrifuged in the cold, resuspended in Hershey-supp, and adjusted to a titer of about 4 X 108/ml. The bacteria were then TABLE
2
BURST SIZE’ OF PHAGE T4Df E. coli HOSTS
ON VARIOUS
Temperature Bacteria
(genotype)
DG76 (wild type) PC2 (dnuC ts mutant) PC3 (dnaG ts mutant)
30”
4o”
88.0 50.0 132.0
81.0 50.0 1.2
’ Similar data were also obtained in several additional experiments. Phage adsorption to these three strains was between 89 and 92% both at 30” and 42”. The transmission coefficient (percentage of infected bacteria yielding at least one phage, e.g., Minner and Bernstein, 1976) was close to 100%.
(12)
separated into two aliquots which were incubated at 30” and 40”. These two aliquots were immediately infected with wild-type phage T4, at a multiplicity of infection of five. After 90 min incubation, phage growth was terminated by addition of chloroform. The progeny phage were then titered using CR63 as host bacteria and standard plating conditions (14). The burst sizes obtained are listed in Table 2. These were calculated as the ratio of the progeny phage titer to the titer of infected bacteria. The burst size in the dn.uG ts mutant infection was more than loo-fold greater at 30” than at 40”, whereas in the other hosts there were no significant differences in phage burst sizes at the two temperatures. These initial burst size measurements prompted a series of DNA synthesis experiments. First, revertants of PC3 (dnuG) were obtained by growing cells at 30” in M9 medium (14) supplemented with Difco Casamino acids, FeCb, and thymine (which we call M9S medium) and 0.1 mg/ml 2aminopurine as a mutagen. The cells were then plated out on EHA agar, supplemented with 20 g/ml thymine and grown at 42”. A revertant colony was picked, reisolated at 42”, and designated PC3R (dnaG). E. coli strains PC2 (dmC), PC3
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COMMUNICATIONS
(dmG), PC3R (dnaG), and DG76 (wild type) were grown at 30’ in M9S to a titer of 2 X lO’/ml, centrifuged in the cold, resuspended in adsorption salts (16), and adjusted to a titer of about 4 X lO’/ml. The temperature was then shifted to 44”. After 5 min, to allow temperature equilibration, phage were added at a multiplicity of infection of about five per cell. At 2 min after infection, after host DNA synthesis is shut off by T4 infection (17), the infected cell culture was diluted threefold in M9S medium which also contained 250 g/ml 2’-deoxyadenosine and 8.7 g/ml [3H]thymidine (0.27 Ci/mmol). At 2-min intervals from 4 to 16 min, then at 20,25,30,40,50, and 60 min, samples of the phage-infected cell cultures were diluted into equal volumes of sampling media (1 .O M KCN, 1.0 mg/ml thymidine) at room temperature, to inhibit further uptake and incorporation of labeled thymidine into bacteriophage DNA. After 15 min O.l-ml portions of diluted samples were spotted onto Whatman 3MM filter papers and dried for 15 min under heat lamps. After drying, the filter papers were washed as described by McCarthy (7), dried under heat lamps, placed in polyethylene vials containing 10 ml Betaphase, and counted for 5 min in a liquid scintillation system, with a tritium energy window. Figure 1 shows that the rates of DNA synthesis of wild-type phage T4D+ were much less in E. cob PC3 (dnaG) than in E. coli DG76 (wild type), PC2 (&I&), and PCSR (dnaG). These results show the necessity of the E. coli dnuG gene product for a normal level of DNA synthesis in wildtype phage T4 infections. As mentioned above, the results shown in Fig. 1 were obtained with cells grown at 44’. Similar experiments were also carried out with cells grown at 40”) 42”, and 43”. With each increase in temperature, the rate of DNA synthesis of wild-type phage T4 in the E. coli dnaG host was found to decrease, while DNA synthesis in the other hosts remained relatively constant (unpublished results). Since both the E. coli dnaG gene product and the phage gene 61 product have RNA primase functions, we assayed the effect
. lW-
“““’
4
’
’
’
a 12 16 20 25 30
I
I
I
40
50
60
MINUTES AFTER INFECTION
FIG. 1. Comparison of DNA synthesis of wild-type phage T4Df on various E. coli hosts. DNA synthesis is measured by the amount of [3H]thymidine (in counts per minute) incorporated into TCA-insoluble material at various times after infection. Experiments were performed at 44’. These data points are the averages from two experiments. Symbols: n , DG76 wild type; A, PC2 dnaC ts mutant; 0, PC3 dmG ts mutant; 0, PC3R revertant of PC3 dmG ts mutant.
on DNA synthesis of a simultaneous defect in both RNA primases. Figure 2, panels A and B, shows the results of measuring the rates of DNA synthesis with wild-type T4D+ and amHL627 (gene 61) phage, grown in parallel on DG76 (wild type), PC3 (dnaG), and PC3R (dnuG) E. coli hosts. Figure 2A shows, as in Fig. 1, that the rate of DNA synthesis of wild-type phage is substantially lower in the dnaG ts mutant host (PC3) than in either wildtype E. coli (DG76) or the revertant of the dnuG ts mutant (PC3R). Figure 2B shows that anHL627 (gene 61) likewise has a lower level of DNA synthesis in a dnuG ts mutant host (PC3) than in either wild-type (DG76) or a revertant of the dnuG ts mutant (PCSR). The rates of DNA synthesis during the exponential period, ks, taken from Fig. 2, are given in Table 3. For the
446
SHORT
COMMUNICATIONS
--
m
20
30
AMHL627 (GENE 58-61)
40 SO 60 m 20 MINUTES AFTER INFECTION
30
40
50
Ml
FIG. 2. Comparison of DNA synthesis of wild-type phage T4D+ to mutant phage amHL627 (gene 61) on various E. coli hosts. Experiments were performed at 44°C. The data from this experiment are typical of data obtained in several additional experiments. Symbols (A and B): w, DG76 wild type; l , PC3 dnaG ts mutant; A, PC3R revertant of PC3 dnaG ts mutant.
three infections involving either a mutant phage, a mutant host, or both a mutant phage and mutant host, the kE values can be expressed as a fraction of the kE for the control infection involving wild-type phage in a wild-type host. These fractional values are listed in the last column of Table 3. It TABLE
3
RATES OF EXPONENTIAL DNA SYNTHESIS Phage
(genotype)
Bacteria (genotype)
kx’
Fractional 6s
T4D+ (wild type)
DG76 (wild type)
0.154
1.00
T4D+ (wild type)
PC3 (dmG)
0.043
0.28
amHL627 (gene 61)
DG76 (wild type)
0.077
0.50
amHL627 (gene 61)
PC3 (dmG)
0.023
0.149
’ kE, the exponential rate of DNA synthesis, was calculated from the equation N/N,, = eke’. When N/ No is set at 2 and t is set equal to the time it takes to double the amount of DNA synthesized, the value of kE is obtained. The doubling times during the period of exponential DNA increase were measured in Fig. 2 and were used to calculate the values of kE for each type of infection.
can be seen that the fractional rate of DNA synthesis in the doubly mutant infection, amHL627 (gene 61) phage in the E. coli dnaG ts mutant, 0.149, is approximately equal to the product of the fractional kEs for the singly mutant infections (0.28 X 0.50 = 0.140). This result would be expected if the two primases act independently of each other. This suggests either that they function at the same step(s) without any interaction between them, or that they function at separate steps. The T4 gene 61 amber mutant expresses partial function (Fig. 1) and has limited viability (Table 1) in the E. coli dnaG mutant host at the restrictive temperature. This suggests that other proteins, such as the E. coli RNA polymerase, may partially substitute for the defective DNA synthesis functions (18) or else that the E. coli dnaG protein is not fully inactivated. A simple hypothesis consistent with these data is that the E. coli dnuG primase is used for initiation at replicative origins whereas the gene 61 primase, as discussed by Liu and Alberts (5), is used in the de novo initiation of Okasaki fragments at the lagging side of the replication fork. This hypothesis fits with the finding that an in vitro system, containing seven purified phage replicative proteins but not including the dnuG protein, is able to carry
SHORT
COMMUNICATIONS
out DNA elongation at a near normal rate and with a near normal level of accuracy (4). This implies that the dmG protein is unlikely to be needed for elongation and is thus more likely to be required for initiation of phage DNA synthesis.
ACKNOWLEDGMENTS I wish to thank H. Bernstein, C. Bernstein, V. Johns, and S. Mufti for their assistance and guidance through these experiments. This work was supported by an NIH Grant GM27219 to H. and C. Bernstein.
REFERENCES 1. WICKNER, S., Annu. Rev. Biochem. 47, 11631191 (1978). 2. KORNBERG, A., “DNA Replication.” Freeman, San Francisco, 1980. 3. BOUCHE, J.-P., ROWEN, L., and KORNBERG, A., J. Biol. Chem. 253, 765-769 (1978). 4. ALBERT& B. M., BARRY, J., BEDINGER, P., BURKE, R. L., HIBNER, U., LIU, C.-C., and SHERIDAN, R., In “Mechanistic Studies of DNA Replication and Genetic Recombination” (B. M. Alberts and C. F. Fox, eds.), pp. 449-484. Academic Press, New York, 1980.
447
5. LIU, C., and ALBERT% B., Proc. Nat. Ad. Sci. USA 77, 5698-5702 (1980). 6. BRESCHKIN, A., and MOSIG, G., J. Mol. Biol. 112, 295-308 (1977). 7. MCCARTHY, D., J. Mol. Biol. 127, 265-283 (1979). 8. LIU, L. F., LIU, C.-C., and ALBERTS, B. M., Nature (London) 281, 456-461 (1979). 9. MCCARTHY, D., MINNER, C., BERNSTEIN, H., and BERNSTEIN, C., J. Mol. Biol. 106, 963-981 (1976). 10. WOLF, B., NEWMAN, A., and GLASER, D., J. Mol. Biol. 32, 611-629 (1968). 11. CARL, P., Mol. Gen. Genet. 109, 10’7-122 (1970). 22. EPSTEIN, R. H., BOLLE, A., STEINBERG, C. M., KELLENBERGER, E., BOY DE LA TOUR, E., CHEVALLEY, R., EDGAR, R. S., DENHARDT, G. H., and LIELAUSIS, A., Cold Spring Harbor Symp. Quunt. Biol. 28, 375-394 (1963). 13. STEINBERG, C. M., and EDGAR, R. S., Genetics 47, 187-208 (1962). 14. ADAMS, M., In “Bacteriophages.” Interscience, London, 1959. 15. MINNER, C. A., and BERNSTEIN, H., J. Gen. Viral. 31,277-280 (1976). 16. SCHNEIDER, S., BERNSTEIN, C., and BERNSTEIN, H., Mol. Gen. Genet. 167, 185-195 (1978). 17. HERSHEY, A. D., DIXON, J., and CHASE, M., J. Gen. Physiol. 36, 777-789 (1953). 18. LUDER, A., and MOSIG, G., Proc. Nat. Acad. Sci. USA 79, 1101-1105 (1981).
123,443-447 (1982)
SHORT COMMUNICATIONS Escherichia
co/i dnaG Gene Product Is Required for a Normal Rate of Phage T4 DNA Synthesis CLAVE LILIEN’
Department
of Molecular and Medical Microbiology, Tucson, Arizona
University 85724
of Arizona College of Medicine,
Received May 25, 1982; accepted August 23, 1982 An Escherichia coli dnaG temperature-sensitive (ts) mutant, which produces a defective RNA primase, does not permit normal bacteriophage T4 growth and a normal rate of DNA synthesis. By contrast an E. coli dnaC ts mutant, a revertant of the dnoG ts mutant, and wild type allow a normal rate of phage T4 DNA synthesis. Evidence is presented that the phage gene 61 RNA primase and the host dnuG primase act independently of each other. We speculate that the host dnaG primase may be utilized for initiation of phage T4 DNA synthesis at replicative origins.
differences suggest that the two prim&es do not perform identical functions. There is evidence that some E. coli host replicative proteins are utilized in phage T4 DNA synthesis (6, 7). Breschkin and Mosig (6) showed that the E. coli dnaG primase is required for the residual primary DNA replication that occurs in a phage T4 gene 32 mutant infection. McCarthy (7), in our laboratory, showed that another E. coli replicative protein, the DNA gyrase, could partially substitute for the products of phage T4 genes 39,52, and 60, which jointly specify a topoisomerase (8). The phage topoisomerase, or substituting host gyrase, appear to be required for a step in initiation of phage DNA synthesis (7,9). The present experiments were undertaken to test whether the E. coli dnaG gene function is required in vivo for phage T4 DNA synthesis. The E. coli strains used in these experiments were PC2 (dnaC ts mutant), PC3 (dnaG ts mutant), PC3R (revertant of dnaG ts mutant), DG76 (wild type), and CR63 (wild type). These are listed in Table 1. PC2 (dnaC ts mutant) is defective in the initiation of replication in E. coli (1 l), and there is no a priori reason to expect it to affect phage T4 DNA replication. With the exception of CR63, these strains were obtained from the E. coli Stock Center, Yale
The product of the E. coli dnaG gene is an RNA primase (1, 2). When singlestranded circular DNA from phage G4 is used in vitro as a template, the dnaG primase synthesizes a 26 to 29 residue RNA transcript at the origin of replication (3). If primase action is coupled directly to replication, the RNA transcript is abbreviated to a few nucleotides by an early extension by DNA polymerase (2). Alberts et al. (4) have presented evidence that the product of phage T4 gene 61 is also an RNA primase that operates together with gene 41 protein, a DNA helicase, to synthesize RNA primers. These primers are pentaribonucleotides of general sequence pppApCpNpNpNp, which, in the presence of the remaining five replicative proteins, are utilized to prime de nouo T4 DNA chain starts during lagging strand synthesis (5). Although the E. coli dnuG gene product and the product of phage T4 gene 61 both have primase activity, they also have several important differences (2). The phage T4 gene 61 primase has single-stranded DNA-dependent ATPase activity, and can utilize only rNTPS. The E. coli dnaG primase does not have such an ATPase activity, and can utilize dNTPs in the presence of rATP, as well as rNTPS. These 1 To whom reprint
requests should be addressed. 443
0042-6822/82/160443-05$02.00/O Copyright All rights
Q 1982 by Academic of reproductionin
Press, Inc. reserved.
any form
444
SHORT
COMMUNICATIONS TABLE
1
BACTERIAL STRAINS Strain
Relevant
genotype
Comments
Source
Reference
DG76
leuB6, thyA47, deoC3, rpsL153, X- sexF-
This strain is the parental type of PC2, PC3, and PC5
E. coli Stock Center
(10)
PC2
lcuB6, thyA47, dnaC2, deoC3, rpsL153, strA157, h- sexF-
dnoC2 is a temperaturesensitive mutant
E. coli Stock Center
(11)
PC3
leuB6, thyA47, dnuG3, deoC3, rpsL153, XserF-
dnaG3 is a temperaturesensitive mutant
E. coli Stock Center
(11)
PCBR
leuB6, thyA47, dnaG3 (rev), deoC3, rpsL153, A- sexF-
A temperatureinsensitive revertant of PC3
Obtained as described in text
CR63
supD60
Standard permissive host for phage T4 am mutants
This laboratory
University, New Haven, Connecticut, through the courtesy of Dr. Barbara Bachmann. The first evidence that E. coli dnaG gene product functions in phage T4 replication was seen in burst size experiments. E. coli cells were grown in Hershey broth (13) supplemented with 20 g/ml thymine (Hershey-supp), to about 2 X 107/ml at 30”, centrifuged in the cold, resuspended in Hershey-supp, and adjusted to a titer of about 4 X 108/ml. The bacteria were then TABLE
2
BURST SIZE’ OF PHAGE T4Df E. coli HOSTS
ON VARIOUS
Temperature Bacteria
(genotype)
DG76 (wild type) PC2 (dnuC ts mutant) PC3 (dnaG ts mutant)
30”
4o”
88.0 50.0 132.0
81.0 50.0 1.2
’ Similar data were also obtained in several additional experiments. Phage adsorption to these three strains was between 89 and 92% both at 30” and 42”. The transmission coefficient (percentage of infected bacteria yielding at least one phage, e.g., Minner and Bernstein, 1976) was close to 100%.
(12)
separated into two aliquots which were incubated at 30” and 40”. These two aliquots were immediately infected with wild-type phage T4, at a multiplicity of infection of five. After 90 min incubation, phage growth was terminated by addition of chloroform. The progeny phage were then titered using CR63 as host bacteria and standard plating conditions (14). The burst sizes obtained are listed in Table 2. These were calculated as the ratio of the progeny phage titer to the titer of infected bacteria. The burst size in the dn.uG ts mutant infection was more than loo-fold greater at 30” than at 40”, whereas in the other hosts there were no significant differences in phage burst sizes at the two temperatures. These initial burst size measurements prompted a series of DNA synthesis experiments. First, revertants of PC3 (dnuG) were obtained by growing cells at 30” in M9 medium (14) supplemented with Difco Casamino acids, FeCb, and thymine (which we call M9S medium) and 0.1 mg/ml 2aminopurine as a mutagen. The cells were then plated out on EHA agar, supplemented with 20 g/ml thymine and grown at 42”. A revertant colony was picked, reisolated at 42”, and designated PC3R (dnaG). E. coli strains PC2 (dmC), PC3
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445
COMMUNICATIONS
(dmG), PC3R (dnaG), and DG76 (wild type) were grown at 30’ in M9S to a titer of 2 X lO’/ml, centrifuged in the cold, resuspended in adsorption salts (16), and adjusted to a titer of about 4 X lO’/ml. The temperature was then shifted to 44”. After 5 min, to allow temperature equilibration, phage were added at a multiplicity of infection of about five per cell. At 2 min after infection, after host DNA synthesis is shut off by T4 infection (17), the infected cell culture was diluted threefold in M9S medium which also contained 250 g/ml 2’-deoxyadenosine and 8.7 g/ml [3H]thymidine (0.27 Ci/mmol). At 2-min intervals from 4 to 16 min, then at 20,25,30,40,50, and 60 min, samples of the phage-infected cell cultures were diluted into equal volumes of sampling media (1 .O M KCN, 1.0 mg/ml thymidine) at room temperature, to inhibit further uptake and incorporation of labeled thymidine into bacteriophage DNA. After 15 min O.l-ml portions of diluted samples were spotted onto Whatman 3MM filter papers and dried for 15 min under heat lamps. After drying, the filter papers were washed as described by McCarthy (7), dried under heat lamps, placed in polyethylene vials containing 10 ml Betaphase, and counted for 5 min in a liquid scintillation system, with a tritium energy window. Figure 1 shows that the rates of DNA synthesis of wild-type phage T4D+ were much less in E. cob PC3 (dnaG) than in E. coli DG76 (wild type), PC2 (&I&), and PCSR (dnaG). These results show the necessity of the E. coli dnuG gene product for a normal level of DNA synthesis in wildtype phage T4 infections. As mentioned above, the results shown in Fig. 1 were obtained with cells grown at 44’. Similar experiments were also carried out with cells grown at 40”) 42”, and 43”. With each increase in temperature, the rate of DNA synthesis of wild-type phage T4 in the E. coli dnaG host was found to decrease, while DNA synthesis in the other hosts remained relatively constant (unpublished results). Since both the E. coli dnaG gene product and the phage gene 61 product have RNA primase functions, we assayed the effect
. lW-
“““’
4
’
’
’
a 12 16 20 25 30
I
I
I
40
50
60
MINUTES AFTER INFECTION
FIG. 1. Comparison of DNA synthesis of wild-type phage T4Df on various E. coli hosts. DNA synthesis is measured by the amount of [3H]thymidine (in counts per minute) incorporated into TCA-insoluble material at various times after infection. Experiments were performed at 44’. These data points are the averages from two experiments. Symbols: n , DG76 wild type; A, PC2 dnaC ts mutant; 0, PC3 dmG ts mutant; 0, PC3R revertant of PC3 dmG ts mutant.
on DNA synthesis of a simultaneous defect in both RNA primases. Figure 2, panels A and B, shows the results of measuring the rates of DNA synthesis with wild-type T4D+ and amHL627 (gene 61) phage, grown in parallel on DG76 (wild type), PC3 (dnaG), and PC3R (dnuG) E. coli hosts. Figure 2A shows, as in Fig. 1, that the rate of DNA synthesis of wild-type phage is substantially lower in the dnaG ts mutant host (PC3) than in either wildtype E. coli (DG76) or the revertant of the dnuG ts mutant (PC3R). Figure 2B shows that anHL627 (gene 61) likewise has a lower level of DNA synthesis in a dnuG ts mutant host (PC3) than in either wild-type (DG76) or a revertant of the dnuG ts mutant (PCSR). The rates of DNA synthesis during the exponential period, ks, taken from Fig. 2, are given in Table 3. For the
446
SHORT
COMMUNICATIONS
--
m
20
30
AMHL627 (GENE 58-61)
40 SO 60 m 20 MINUTES AFTER INFECTION
30
40
50
Ml
FIG. 2. Comparison of DNA synthesis of wild-type phage T4D+ to mutant phage amHL627 (gene 61) on various E. coli hosts. Experiments were performed at 44°C. The data from this experiment are typical of data obtained in several additional experiments. Symbols (A and B): w, DG76 wild type; l , PC3 dnaG ts mutant; A, PC3R revertant of PC3 dnaG ts mutant.
three infections involving either a mutant phage, a mutant host, or both a mutant phage and mutant host, the kE values can be expressed as a fraction of the kE for the control infection involving wild-type phage in a wild-type host. These fractional values are listed in the last column of Table 3. It TABLE
3
RATES OF EXPONENTIAL DNA SYNTHESIS Phage
(genotype)
Bacteria (genotype)
kx’
Fractional 6s
T4D+ (wild type)
DG76 (wild type)
0.154
1.00
T4D+ (wild type)
PC3 (dmG)
0.043
0.28
amHL627 (gene 61)
DG76 (wild type)
0.077
0.50
amHL627 (gene 61)
PC3 (dmG)
0.023
0.149
’ kE, the exponential rate of DNA synthesis, was calculated from the equation N/N,, = eke’. When N/ No is set at 2 and t is set equal to the time it takes to double the amount of DNA synthesized, the value of kE is obtained. The doubling times during the period of exponential DNA increase were measured in Fig. 2 and were used to calculate the values of kE for each type of infection.
can be seen that the fractional rate of DNA synthesis in the doubly mutant infection, amHL627 (gene 61) phage in the E. coli dnaG ts mutant, 0.149, is approximately equal to the product of the fractional kEs for the singly mutant infections (0.28 X 0.50 = 0.140). This result would be expected if the two primases act independently of each other. This suggests either that they function at the same step(s) without any interaction between them, or that they function at separate steps. The T4 gene 61 amber mutant expresses partial function (Fig. 1) and has limited viability (Table 1) in the E. coli dnaG mutant host at the restrictive temperature. This suggests that other proteins, such as the E. coli RNA polymerase, may partially substitute for the defective DNA synthesis functions (18) or else that the E. coli dnaG protein is not fully inactivated. A simple hypothesis consistent with these data is that the E. coli dnuG primase is used for initiation at replicative origins whereas the gene 61 primase, as discussed by Liu and Alberts (5), is used in the de novo initiation of Okasaki fragments at the lagging side of the replication fork. This hypothesis fits with the finding that an in vitro system, containing seven purified phage replicative proteins but not including the dnuG protein, is able to carry
SHORT
COMMUNICATIONS
out DNA elongation at a near normal rate and with a near normal level of accuracy (4). This implies that the dmG protein is unlikely to be needed for elongation and is thus more likely to be required for initiation of phage DNA synthesis.
ACKNOWLEDGMENTS I wish to thank H. Bernstein, C. Bernstein, V. Johns, and S. Mufti for their assistance and guidance through these experiments. This work was supported by an NIH Grant GM27219 to H. and C. Bernstein.
REFERENCES 1. WICKNER, S., Annu. Rev. Biochem. 47, 11631191 (1978). 2. KORNBERG, A., “DNA Replication.” Freeman, San Francisco, 1980. 3. BOUCHE, J.-P., ROWEN, L., and KORNBERG, A., J. Biol. Chem. 253, 765-769 (1978). 4. ALBERT& B. M., BARRY, J., BEDINGER, P., BURKE, R. L., HIBNER, U., LIU, C.-C., and SHERIDAN, R., In “Mechanistic Studies of DNA Replication and Genetic Recombination” (B. M. Alberts and C. F. Fox, eds.), pp. 449-484. Academic Press, New York, 1980.
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