VIROLOGY

96, 642-645 (1979)

Incorporation

of Thymine

KENNETH Department

of Biology,

into +e DNA during

S. LOVEDAY’

Massachusetts

AND

MAURICE

Institute of Technology, Accepted April

of Bacillus subtilis

Transfection

S. FOX

Cambridge,

Massachusetts

02139

18, 1979

Phage $e, whose DNA contains hydroxymethyl uracil in place of thymine, excludes thymine from its DNA by inducing enzymes that degrade TTP and block thymidylate synthetase. During transfection of Bacillus subtilis with 4e DNA, however, progeny phage can be labeled with [3H]thymine at an average of 1000 thymine residues per phage, a level eightfold higher than that found after normal phage infection. This observation supports a transfection model in which bacterial polymerases, using thymine, repair gaps in phage DNA molecules created by the renaturation of single-strand fragments of phage DNA taken up by competent cells.

For the majority of Bacillus subtilis phages, the number of infective centers formed by transfecting DNA increases with the second or third power of DNA concentration, suggesting that several DNA molecules are required to form an infective center (1-5). Previous investigators have suggested that phage DNA is damaged during or after entry into the cell and that several DNA molecules (taken up independently) must recombine to form an intact phage genome (3, 6-8). Our observations describing the fate of +e transfecting DNA in B. subtilis (9) suggest a different model. Double-stranded transfecting DNA (9), like bacterial transforming DNA (11-14), is rendered single stranded during uptake by competent B. subtilis bacteria. Complementary fragments of single-stranded phage DNA can then anneal within the cell. Bacterial polymerases can repair gaps in such molecules. In those cases in which a complete doublestranded phage genome has been assembled, an infective center results. This proposal accounts for the multipower dependence of infective center formation on phage DNA concentration and the abundance of recombinants among the progeny since uptake from several phage DNA molecules would be required to provide the complementary single-stranded fragments neces1 To whom reprint requests should be addressed.

0042~6822/79/100642-04$02.00/0 Copyright AU rights

0 19’79 by Academic Press, Inc. of reproduction in any form reserved.

sary for the assembly of a complete genome. Porter and Guild have suggested a similar model to describe transfection in pneumococcus (10). The DNA of +e, like SPOl and SP82, contains hydroxymethyl uracil (HMU) in place of thymine (6,15, IS). After infection, induced phage-coded enzymes that degrade TTP and block thymidylate synthetase (15) are responsible for the exclusion of thymine from DNA. By using a phage mutant deficient in TTPase, Marcus and Newlon showed that thymine could replace as much as 20% of the HMU in the phage DNA without affecting viability (17’). In the proposed early stage of transfection, bacterial polymerases would be expected to repair the gaps present in renatured phage DNA, using thymine-containing precursors, since the phage functions necessary for the synthesis of HMU would presumably be absent until a more or less complete double-stranded genome had been assembled. The sensitivity of phage multiplication following transfection to inhibition by 6-(p-hydroxyphenylazo)-uracil (HPUra) (9), a specific inhibitor of bacterial DNA synthesis that does not interfere with +e DNA synthesis (18, 19), provides support for this hypothesis. This interpretation suggests a role for polymerase III (the polymerase inhibited by HPUra) in the repair of gaps in the renatured transfecting DNA.

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The incorporation of [3H]thymine into phage DNA that had been taken up during transfection provided further support for this interpretation (9). The observations that will be described show that the [3H]thymine found in phage DNA is also present in the phage that are matured following transfection with +e DNA, thus showing that the phage DNA molecules that had incorporated thymine are in fact intermediates in transfection. The techniques for growing competent B. subtilis strain 168 thy- trp-, for fractionating competent cultures on renogra6n gradients, and for performing transfection experiments with DNA isolated from phage +e have been previously described (9). In order to maximize the transfection yield, a saturating concentration of phage DNA (5 pg/ml) was added to competent cells that had been previously exposed to uv-irradiated bacterial DNA (a procedure that increases the yield of infective centers) (9, 20, 27). After 50 min of exposure to phage DNA in the presence of 100 @i/ml [3H]thymine, the cells were washed twice to remove the unincorporated [3H]thymine and resuspended in a lo-fold greater volume to limit readsorption of any matured phage. After additional incubation for 70 min, the cells were concentrated and lysed by the addition of 250 pg/ml lysozyme. The lysate was centrifuged to equilibrium in a CsCl gradient to purify the phage. Part of the competent culture was infected with intact phage particles to determine the level of [3H]thymine incorporation into the progeny phage during normal infection. A modest level of [3H]thymine incorporation into the progeny phage was observed following normal infection (Fig. 1A). The specific activity based on fractions 14 and 15 is 2.4 x lo+ cpm/phage and is comparable to that previously reported by Roscoe (2.2). Based on a genome size of 120 x lo6 daltons and random incorporation of thymine, this represents an average of about 130 thymine

FIG. 1. Equilibrium centrifugation of phage r$e from infected and transfected cells. The cells in 100 ml competent culture (168 thy-trp-), in GM1 medium containing 4 pggiml thymine, were fractionated using a renografm gradient (9). The lighter fraction (enriched for competent cells) was resuspended in a fmal volume of 8 ml in medium containing no thymine. To enhance the level of transfection, bacterial DNA irradiated for 10 min at 35 ergs/mm*/sec was added (0.2 pg/ml) (7, 9). After 5 min of incubation at 37”, [3H]thymine was added to a final concentration of 100 &i/ml with a specific activity of 125 @.Zi/pg thymine. After an additional 5 min of incubation, the culture was divided into two parts. One part received +e phage at a multiplicity of infection of 5 while the other part received 4e DNA at a final concentration of 5 pg/ml. (A) The phage infected cells lysed after a I-hr incubation at 37” (burst size of 50) and the phage were purified by a CsCl step gradient and a CsCl equilibrium gradient. The data from the equilibrium gradient are plotted in (A). Total radioactivity, 0; total phage, 0. (B) After exposure to transfecting DNA for 50 min at 37”, the cells were washed twice and resuspended in 50 ml of medium containing 4 pg/ml thymine. After a further 70 min of incubation, the cells were centrifuged, resuspended in 3 ml of medium, and lysed by the addition of 250 pg/ml lysozyme (egg white, Sigma). The lysate (burst size of 100) was centrifuged to equilibrium in CsCl to purify the phage. Drops were collected from the bottom of the tube (9), and the fractions containing the phage were pooled and rerun to eliminate the large amount of dispersed radioactivity present in the first equilibrium gradient. The data from the second CsCl gradient are In (B), the specific activity is based on fraction 20 plotted in (B). In (A) the specific activity of the phage (1.9 x 1O-5 cpm/phage, subtracting the background is based on fractions 14 and 15 (2.4 x 10m6cpm/phage). radioactivity of 800 cpm).

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residueslphage genome. The specific activity of phage released by lysis of the transfected cells is eight times higher than the control (1.9 x 10m5cpm/phage) (Fig. 1B). Assuming that the thymine is distributed uniformly among the particles, this would represent about 1000 thymine residues per progeny phage, an average substitution of 1% of the HMU. Within the framework of the model that we have suggested, only the parental transfecting DNA that renatures would be labeled with [3H]thymine. On this basis, we can estimate the expected specific activity of the matured phage. The maximum possible substitution of thymine would be about 50% for each full-length molecule (when two half-sized single-strand pieces of DNA overlapped enough at the middle to renature). With lo5 HMU residues/genome, 50% substitution with thymine (125 &iIpg) would give 1 x lop3 cpm/genome, based on a counting efficiency of 40%. With saturating concentrations of DNA, about 10 phage genome equivalents of DNA are taken up by each competent cell (and presumably each infective center) (9; unpublished results). Although only one complete genome/ cell would have to be reassembled to produce an infective center, it is likely that many additional fragments of phage DNA of variable length would also be reassembled and repaired with [3H]thymine, within the same bacterium. Following phage infection with +e, about 30% of the parental DNA is subsequently packaged and released in progeny phage (17’). It seems reasonable to assume that in this transfection experiment, about 30% of the repaired, parental transfecting DNA molecules (about 10 phage DNA equivalents) would also be packaged into mature phage. If so, there should be an average of three labeled genomes released as phage from each infective center. Since the burst size in the experiment was 100, that would yield a maximum of three labeled genomes/lOO phage x 10m3 cpmllabeled genome or 3 x 10V5cpm/phage. The observed specific activity of 1.9 x low5 cpm/phage is somewhat smaller than the

calculated maximum of 3 x 10w5. The assumption of the minimum hybrid overlap resulting in 50% repair synthesis is of course extreme. Modest regions of overlap would reduce the calculated expectation of thymine incorporation. The fact that the observed value is as high as it is suggests that the average amount of repair for each renatured molecule of transfecting DNA may be substantial. Even with the assumption that there were 20 genome equivalents of transfecting DNA taken up by each infective center, these considerations would suggest 15% repair synthesis in each renatured molecule. The earlier observation of [3H]thymine incorporation into phage DNA following transfection (9) coupled with the present observation lend considerable support to the suggestion that the double-stranded DNA that is taken up in transfection is reduced to single-strand fragments that can reassemble by hybridization of complementary sequences and are repaired by bacterial polymerases using thymine, to form double-strand DNA. In those cells in which a full-length phage genome has been reassembled, an infective center would result. ACKNOWLEDGMENTS This work was supported by Public Health Service Grant AI05388 from the National Institute of Allergy and Infectious Diseases. REFERENCES 1. FISCHBACH,

2. 3. 4. 5. 6. 7. 8. 9.

K., SPATZ, H., and KLOTZ, G., Mol. Gen. Genet. 141, 121-145 (1975). FLOCK, J., and RUTBERG, L., Mol. Gen. Genet. 131, 301-311 (1974). GREEN, D. M., J. Mol. Biol. 22, 1-13 (1966). OKUBO, S., STRAUSS, B., and STODOLSKY, M., Virology 24, 552-562 (1964). SPATZ, H., and TRAUTNER, T., Mol. Gen. Gerwt. 109, 84-106 (19’70). GREEN, D. M., J. Mol. Biol. 10, 438-451 (1964). EPSTEIN, H., J. Viral. 2, 710-715 (1968). SPATZ, H., and TRAUTNER, T., Mol. Gen. Genet. 113, 174-190 (1971). LOVEDAY, K. S., and Fox, M. S., Virology 85, 387-403 (1978).

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10. PORTER, R., and GUILD, W., J. Viral. 25, 60-72 (1978). 11. DAVIWFF-ABELSON, R., and DUBNAU, D., J. Bacterial. 116, 146-153 (1973). 1.2. PIECHOWSKA, M., and FOX, M. S., J. Bacterial. 108, 681-689 (1971). 1s. PIECHOWSKA, M., SOLTYK, A., and SHUGAR, D., J. Bacttiol. 122, 610-622 (1975). 14. SOLTYK, A., SHUGAR, D., and PIECHOWSKA, M., J. Bacterial. 124, 1429-1438 (1975). 15. ROSCOE, D., and TUCKER, R., Virology 29, 157- 166 (1966).

16. YEHLE, C. O., and GANESAN, A. T., J. Viral. 9, 263-272 (1972). 17. MARCUS, M., and NEWLON, 44, 83-93 (1971). 18. BROWN, N., Proc. Nat. 1454-1461 (1970). 19. BROWN, N., J. Mol. Biol.

M. C., Virology

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59, 1-16 (1971).

20. ARWERT, F., and VENEMA, G., Mol. Gen. Genet. 128, 55-72 (1974). 21. EPSTEIN, H., J. Vilirol. 2, 710-715 (1968). 22. ROSCOE, D. H., Virology 38, 527-537 (1969).

Incorporation of thymine into phi e DNA during transfection of Bacillus subtilis.

VIROLOGY 96, 642-645 (1979) Incorporation of Thymine KENNETH Department of Biology, into +e DNA during S. LOVEDAY’ Massachusetts AND MAURICE...
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