JOURNAL OF VIROLOGY, Feb. 1977, p. 806-809 Copyright © 1977 American Society for Microbiology

Vol. 21, No. 2 Printed in U.S.A.

Gene A Protein of Bacteriophage S13 Is Required for SingleStranded DNA Synthesis ETHEL S. TESSMAN* AND PATRICIA K. PETERSON Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907

Received for publication 15 July 1976

The product of gene A of the small icosahedral DNA phage S13 has been found to be needed for single-stranded DNA synthesis in vivo in addition to its previously known role in progeny replicative-form DNA synthesis. It has been known for many years that the gene A protein of the small related icosahedral DNA phages S13 and 4X174 is needed early in infection to initiate progeny replicative-form (RF) synthesis (11, 14, 16). It was later shown that the A protein was a nickase (3, 4, 9) that nicks the viral strand of supercoiled parental RF. The filamentous small DNA phages M13 and fd share many characteristics of DNA replication with the icosahedral small DNA phages, though they differ greatly in other aspects of development (2). For these phages a protein that is analogous to the 4X174 gene A protein, namely the gene 2 protein, is needed for both progeny RF synthesis and singlestranded DNA (SS) synthesis (10, 18). Therefore, the work with gene 2 mutants suggested that gene A protein might be required for SS synthesis of the icosahedral phages. Henry and Knippers (9) predicted that the A protein function would be needed for all stages of DNA replication of 4X174 on the basis of their finding that purified A protein cleaves both RF and SS. Another reason for predicting this late function of the A protein is that a complex which appears to -be composed of rep protein plus the phage-specified F and A proteins is required late in development of OX174 (17). We report here that the gene A product is required for SS DNA synthesis of phage S13. After this work had been completed, studies reaching the same conclusion were reported by Fujisawa and Hayashi (5, 6) for the closely related phage OX174. Our experiments were made possible by finding a temperature-sensitive (ts) mutant in gene A whose cut-off temperature, 41.5°C, is low enough that SS synthesis by wild-type phage is not reduced. This A mutant, tsA1072, was isolated by Ron Baker. Since mutations in most of the genes of S13 and 4X174 can block SS DNA synthesis (11, 16), it was necessary to prove that tsA1072 is mutated only in gene A. This S13 mutant was tested in Escherichia coli Cla

at 41.5°C for complementation with S13 mutants of all genes except gene D, for which no S13 mutants have been found. tsA1072 complemented mutants of all S13 genes except A (Table 1) and therefore is a gene A mutant, showing the expected poor rescue that is characteristic of gene A mutants. There was no complementation with the OX mutant amD56, but S13 mutants in several genes fail to complement 4XD mutants (Tessman, unpublished data) so this lack of complementation is not evidence that tsA1072 carries a mutation in gene D. Evidence that tsA1072 has no temperature-sensitive mutation in gene D comes from two sources, reversion frequency of the mutant and genetic mapping. The reversion frequency of tsA1072 to ts+ is 10-5, which is appropriate for a single mutant and several orders of magnitude too high for a double mutant. Genetic crosses were performed in the suppressing strain C520, and the recombination frequencies shown in Fig. 1 indicate that tsA1072 carries no temperature-sensitive mutation in the vicinity of gene D. We conclude that tsA1072 is a single mutant in gene A. The control phage used in the following shiftup experiments was derived by spontaneous reversion from the double mutant tsA1072amE15 (E is the lysis gene). Thus the control phage differs from the mutant phage only in its temperature sensitivity; the control phage shares with the mutant any cryptic mutations that might be present. To determine whether the gene A protein is needed for SS synthesis, parallel cultures of E. coli C-1412 (1) were infected with either the mutant or the control phage at 350C and shifted up to the nonpernissive temperature, 41.50C, at a time when SS synthesis was well under way (data not shown). Identification of the SS peak in extracts from S13-infected or OX-infected cultures labeled for a long time late in infection was unambiguous because the SS peak was always much larger than the RFI and 806

VOL. 21, 1977 TABLE 1. Complementation of tsA1072: a burst sizes in. mixed infections between tsA1072 and standard phage mutants Standard mutant Burst size of tsA Total burst size 5 amA105 0.24 0.3 amB129 0.04 1.3 tsC17 _b 1.8 amD56 0.3 4 amE15 0.04 10.0 tO amF28 0.06 4.2 amG43 0.02 2.2 amH66 0.03 2.3 aComplementation was in Cla at 41.50C as deE scribed previously (15). All phages were S13 muo tants except for the D mutant, which is a OX174 mutant. The self-bursts were: tsA1072, 0.15; amB129, 0.01; tsC17, 0.01; OXamD56, 0.10; amE15, 2 0.02; amF28, 0.15; amG43, 0.17; amH66, 0.12.

tsA

-

-

X3LIIo *

350 41.5

RFI

RFI

B C

A

omA113

L

2

b -, Burst size could not be measured.

Gene

807

NOTES

tsA1072

D E tsC17 amE15

I~~~~~~~~~~ 2.7 2.6 2.0

,,

20

Fraction no.

4.8

_4

I

5.1

5.7 X 10

Recombination frequency FIG. 1. Genetic mapping of tsA1072 by two-factor crosses between tsA1072 and mutants of genes A, C, and E ofphage S13. The figure shows that tsA1072 has no ts mutation in gene D. Recombination *e *ecearexpressed as number of wild-type phage que.eies are particles divided by the total number of progeny particles. The frequencies shown are all in units of 10-4. Crosses were carried out by mixedly infecting broth cultures of the suppressing strain E. coli C-520, diluting l03fold after adsorption; and plating, after burst, on E. coli C at 42.0°C for wild-type recombinants. Total burst was plated on C-520.

RFII peaks (17). Labeling at late times with long pulses gave gradients showing either very small or undetectable amounts of RF. In Fig. 2 and 3, the SS peak was at fraction 28. The fact that the R' peak was at fraction 23 is known from nnumerous previous from Fig. 2 and also rrm experiments with RF that that used the same sedi experiments with sedimentation conditions. Previous experiments also showed that RFII appears at fractions 18 and 19.

R2useo usae

FIG. 2. Lack offormation of SS DNA by tsA1072 after shift-up to high temperature. E. coli C-1412 (1) was grown in M9 medium (13)-0.05% Casamino Acids at 37°C to 2 x 10" cellslml, and MgSO4 was added to 2 X 10-2 M. The cells were infected with tsA1072-amE at a multiplicity of infection of 5, left at 35C for 37 mm. At this time, 5 ml of infected culture was transferred to 41.5°C, while 5 ml remained at 350C. Aeration was continued for both cultures. At 49 min postinfection, [3H]thymidine 15 Ci/mmol) was added each (AmershamiSearle; culture to give 200 ptCi/ml. The cultures were to chilled T . at 69 mn, centrifuged, gently lysed, and sedimented through a 5 to 20% neutral sucrose high-salt gradient as described by Francke and Ray (3), except that the gently lysed extracts were heated at 600C in sodium dodecyl sulfate for 15 mm before the addition of Pronase, and Pronase treatment was carried out only for 30 min at 3r7C. Centrifugation was for 17.5 h at 25,000 rpm at 4°C in the SW27 rotor ofa Beckman L50 centrifuge. One-milliliter fractions were collected from the top of the tube. Samples (02 ml) were added to 5 ml of a scintillation fluid composed of 80 parts water, 100 ml ofAquasol, and 0.5 parts glacial acetic acid. The peak at fraction 28 is SS DNA (see text). present, is known to peak at fraction 23 RFI, when from the parallel gradient done in the same run .

shown in Fig. 3 and from numerous previous experiments with RF using the same sedimentation conditions. RFII is known to peak at fractions 18 and 19 from these same experiments.

808

J. VIROL.

NOTES

25h

18 16

S13 is needed for single-stranded DNA synthesis. This result fulfills the prediction of Henry and . 1 L Knippers (9) that the A product functions + SS synthesis as well as in progeny RF syntsinthesis. fr r They hypothesized that one of the functions of the A product was the cleaving of single-stranded DNA into the unit-length pieces that would be enclosed in mature phage parti-

A

cles. TSince the A product acts both early and late O 14 in infection, the DNA substrate that it nicks might be expected to be structurally the same _l x i2 35 the same and late, as welltheasearly having both | base early sequence. However, E substrate is CL 41.50 * supercoiled RF (3, 4) and the late substrate is a _.l.l 0 _ relaxed RF molecule with a single-stranded tail, a "sigma" structure (MacHattie, cited by Denhardt [2]). This is the structure predicted 8 RFI by the rolling-circle model of Gilbert and Dressler The work of the many authors 6 whose this structure as the precursor of data (7). support DNA is reviewed Denhardt The most (2). by *5ss _ I d \ 44 recent data indicating such a structure, are \l those of Fujisawa and Hayashi (5). The question of the structural identity of the early and late substrates for the A protein was dealt with I by Henry and Knippers (9) when they sought to 30 20 explain their findings that the A protein could nick ¢X174 RF andthat could cleave 4X174 Fraction no. cleavesSS.a postulated thealso A protein They FIG. 3. Enhanced formation of SS DNA by the hairpin-like single-stranded structure, which -

Z

0)

-

41.

-

-

control phage ts+-amE after shift-up to high temperature. Procedures were as described in the legend to Fig. 2. The peak at fraction 28 is SS DNA. Note the

in supercoiled RF is assumed to "flip out" from the double helix. Experimental evidence for the existence of hairpin structures in supercoiled

difference in scale between Fig. 2 and 3.

RF ofOX174 is cited by Henry and Knippers (9). In the single-stranded tail of the SS precursor molecule such a hairpin structure could also

At 37 min postinfection, samples of the tsAinfected culture and the ts+-infected culture occur. The requirement for the A product in SS were shifted to 41.5°C and shaking was continued. [3H]thymidine was added to 200 juCi/ml at synthesis may explain why much more A pro49 min after infection, and the cultures were tein is made than is needed for RF synthesis chilled at 69 min after infection. Cold thymi- alone. Addition of chloramphenicol to 30 ,g/ml dine was added to 200 ug/ml at the time of very much reduces A protein synthesis (8) yet chilling. The infected cultures were then centri- does not cause much reduction in progeny RF fuged, gently lysed, and sedimented according synthesis (16). Therefore, it had previously to the procedures of Francke and Ray (3), ex- seemed that A protein was made in much cept for the modification described in the legend greater amounts than was needed for its known to Fig. 2. The shift-up of the tsA-infected cul- early function. Neither the present experiments nor the exture to 41.5°C resulted in a drastic decrease in SS synthesis compared with the parallel cul- periments of Fujisawa and Hayashi (6) distinture that had remained at 350C (Fig. 2). In guish whether it is the A or the A* protein (A* contrast, shift-up of the ts+-infected culture to is the shorter product ofthe A gene [12]) or both 41.50C showed threefold more synthesis of SS at that are needed in SS synthesis. The experi41.5°C than did the parallel ts+ culture at 350C ments of Henry and Knippers (9) suggest that (Fig. 3). It should be noted that the scales of the at least the A protein is required for SS syntheordinates of Fig. 2 and 3 are different. The sis. 5 synthesis by the tsA mutant was decrease in,This work was supported by Public Health Service grant about 21-fold. AI-11853 from the National Institute of Allergy and InfecWe conclude that the gene A product of phage tious Diseases.

NOTES

VOL. 21, 1977 We thank Irwin Tessman for his comments on the manuscript.

LITERATURE CITED 1. Calendar, R., B. Lindqvist, G. Sironi, and A. J. Clark. 1970. Characterization of REP mutants and their interaction with P2 phage. Virology 40:72-83. 2. Denhardt, D. T. 1975. The single-stranded DNA phages. CRC Crit. Rev. Microbiol. 4:161-223. 3. Francke, B., and D. S. Ray. 1971. Formation of the parental replicative form DNA of bacteriophage 4X174 and initial events in its replication. J. Mol. Biol. 61:565-586. 4. Francke, B., and D. S. Ray. 1972. Cis-limited action of the gene A product of bacteriophage 4X174 and the essential bacterial site. Proc. Natl. Acad. Sci. U.S.A. 69:475-479. 5. Fujisawa, H., and M. Hayashi. 1976. Viral DNA-synthesizing intermediate complex isolated during assembly of bacteriophage X174. J. Virol. 19:409-415. 6. Fujisawa, H., and M. Hayashi. 1976. Gene A product of 4X174 is required for site-specific endonucleolytic cleavage during single-stranded DNA synthesis in vivo. J. Virol. 19:416-424. 7. Gilbert, W., and D. Dressier. 1968. DNA replication: the rolling circle model. Cold Spring Harbor Symp. Quant. Biol. 33:473-484. 8. Godson, G. N. 1971. 4X174 gene expression in UVirradiated cells treated with chloramphenicol. Virology 45:788-792.

809

9. Henry, T. J., and R. Knippers. 1974. Isolation and function of the gene A initiator of 4X174, a highly specific DNA endonuclease. Proc. Natl. Acad. Sci. U.S.A.

10. 11.

12.

13. 14.

15. 16.

71:1M59-1553.

Lin, N. S.-C., and D. Pratt. 1972. Role of bacteriophage M13 in viral DNA replication. J. Mol. Biol. 72:37-49. Lindqvist, B. H., and R. L. Sinsheimer. 1967. The process of infection with bacteriophage 4X174. XV. Bacteriophage DNA synthesis in abortive infections with a set of conditional lethal mutants. J. Mol. 30:69-80. Linney, E. A., M. N. Hayashi, and M. Hayashi. 1972. Gene A of 4X174. Virology 50:381-387. Miller, J. H. 1972. Experiments in molecular genetics, p. 431. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Shleser, R., A. Puga, and E. S. Tessman. 1969. Synthesis of replicative form deoxyribonucleic acid and messenger ribonucleic acid by gene IV mutants of bacteriophage S13.J. Virol. 4:394-399. Tesman, E. S. 1965. Complementation groups in phage S13. Virology 25:303-321. Tessman, E. S. 1966. Mutants of bacteriophage S13

blocked in infectious DNA synthesis. J. Mol. Biol. 17:218-236. 17. Tessman, E. S., and P. K. Peterson, 1976. Bacteriol rep- mutations that block development of small DNA bacteriophages late in infection. J. Virol. 20:400-412. 18. Tseng, B., and D. Marvin. 1972. Filamentous bacterial viruses. VI. Role of fd gene 2 in deoxyribonucleic acid replication. J. Virol. 10:384-391.

Gene A protein of bacteriophage S13 is required for singel-stranded DNA synthesis.

JOURNAL OF VIROLOGY, Feb. 1977, p. 806-809 Copyright © 1977 American Society for Microbiology Vol. 21, No. 2 Printed in U.S.A. Gene A Protein of Bac...
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