Vol. 22, No. 3 Printed in U.S.A.
JOURNAL OF VIROLOGY, June 1977, p. 835-838 Copyright © 1977 American Society for Microbiology
Metabolism of Uracil-Containing DNA: Degradation of Bacteriophage PBS2 DNA in Bacillus subtilis1 BRUCE K. DUNCAN AND HUBER R. WARNER* Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55108
Received for publication 28 December 1976
When Bacillus subtilis is infected by the uracil-containing DNA phage PBS2, the parental DNA labeled with radioactive uracil and cytosine remains acid insoluble. If the synthesis of the phage-induced uracil-DNA N-glycosidase inhibitor is prevented, the parental DNA is completely degraded to acid-soluble products beginning at about 6 min after infection. The host N-glycosidase probably initiates the degradation pathway, with nucleases being responsible for the remaining degradation of the DNA.
Although the DNA in almost all biological systems contains thymine, a consideration of the known pathways necessary for DNA synthesis suggests that the occasional incorporation of uracil in place of thymine in DNA is at least a possibility (13). Bessman et al. have shown that dUTP is a substrate for Escherichia coli DNA polymerase I in vitro (1), and presumably dUTP could be used by other DNA polymerases as well. In addition, the spontaneous or chemically induced deamination of cytosine in situ would also result in the presence of uracil in DNA and occurs at biologically significant rates (2). The presence of adenine-uracil base pairs in DNA could conceivably cause defective DNA function, whereas the deamination of cytosine to uracil would induce mutations. An enzyme that could play an obvious role in repair of such uracil-containing DNA regions has recently been discovered in E. coli (7) and Bacillus subtilis (4). This enzyme is a uracil-DNA N-glycosidase, which catalyzes the following reaction: DNA -- uracil + apyrimidinic DNA. Thus, uracil can be removed from DNA, and the DNA can be repaired by the sequential action of endonuclease II, DNA polymerase I, and DNA ligase (8). The B. subtilis phage PBS2 is a virus whose DNA contains uracil completely substituted for thymine (12). Several proteins are induced by this phage to facilitate the synthesis of uracilcontaining DNA in cells normally engaged in the synthesis of thymine-containing DNA (3-5, 9-11). One of these proteins is an inhibitor of the B. subtilis uracil-DNA N-glycosidase. Inhibition of this enzyme commences within 1 min 1 Scientific Paper no. 9764 of the Minnesota Agricultural Experiment Station.
after infection and is complete by about 5 min after infection (3-5). We have used this virus system to study the stability of uracil-containing DNA in bacterial cells. Radioactive PBS2 phage were prepared by adding [5-3H]uracil (39 ,uCi/ml; 11 Ci/mmol) to a B. subtilis SB19 culture in TYG medium (5) 5 min after infection with PBS2 and then shaking the culture at 370C for 100 min. The phage were purified from the lysate by CsCl gradient centrifugation and had a specific activity of 6.1 x 104 cpm/10'0 PFU. Both cytosine and uracil are labeled by [5-3H]uracil in this manner (75% of the radioactivity is in cytosine and 25% is in uracil). These radioactive phage were then used to study the stability of uracil-containing DNA in B. subtilis cells under a variety of conditions. When B. subtilis was infected with radioactive phage at a multiplicity of infection of about 0.3, little acid solubilization of the DNA occurred after infection (Fig. 1A). However, when actinomycin D was added to the culture 2 min prior to infection to prevent induction of phageinduced proteins, including the uracil-DNA Nglycosidase inhibitor, rapid and complete acid solubilization of the phage DNA occurred. A similar addition of chloramphenicol also caused acid solubilization of the DNA (Fig. 1B). When the chloramphenicol was added 6 min after the phage, the N-glycosidase should have already been fully inhibited (4, 5), and no significant acid-solubilization was observed. The acid-soluble products were characterized in two ways. Portions (0.75 ml) of the trichloroacetic acid supernatant fractions were neutralized with 0.225 ml of 1 M Tris base, and 0.25-ml portions of these neutralized samples were diluted with 1 ml of water and chromatographed on columns (0.3 by 5.0 cm) of Bio-Rad AG-1-
835
836
NOTES
J. VIROL.
formate anion-exchange resin. Neutral and positively charged compounds were eluted with water, and nucleoside monophosphates were eluted with 0.2 M ammonium formate, pH 5. The amount of radioactivity eluted in the water wash is shown in Fig. 1. Almost no radioactivity was eluted with the monophosphate fraction (data not shown). These results indicate that neutral and positively charged molecules constitute 65 to 95% of the acid-soluble fraction, and that acid-soluble oligonucleotides constitute most of the remainder. The acid-soluble products were further characterized by chromatography of 0.7-ml portions of the neutralized samples on Bio-Gel P-2 columns. These columns had been previously found to be useful for the separation of dTMP, 100%---
A
ACTINOMYCIN
thymidine, and thymine (14). Elution profiles of the 15-min samples are shown in Fig. 2. Small amounts of radioactive material elute in the positions corresponding to oligonucleotides (fractions 12 to 17), mononucleotides (fractions 18 to 20), and uracil (fractions 32 to 37). The major radioactive product (fractions 28 to 30) did not elute as an identifiable pyrimidine derivative. Lyophilization showed that this product is volatile, suggesting that it is 3H2O. We confirmed that 3H2O does elute in fractions 28 to 30 by chromatographing authentic 3H20 along with dUMP and uracil (Fig. 2C). Under the conditions used here, uracil is retarded bd1.5
B 100%---CHLORAMPHENICOL
15 min
'.5~~~~~~~~~~~. 0~~~~~~~~~~~~~~~. .0~~~~~~~~~~~~~ E > B. ACTINOMYC~~~bI - 15 min ~1.0
i15 X~~~~~
FG 1.0
A. CHLORAMPHENICOL -
*
A
0.-
0.
LL B. ACTINOMYCIN CL
E
-15 min
.0
1 .0STN-D 1.0
~~~~~~~~~~C
.
CONTROL 0
10
20
0 30 20 10 MINUTES AFTER INFECTION
Q5>
30
FIG. 1. Acid solubilization of PBS2 DNA after infection ofB. subtilis. B. subtilis SB19 was grown to an absorbency at 650 nm of 0.7 in TYG medium at 37'C. Actinomycin D or chloramphenicol was added 2 min before the radioactive phage, 6 min after the phage, or not at all. The final concentrations of actinomycin D and chloramphenicol, when added, were 20 and 100 pglml, respectively. At various times, 1ml samples were removed, added to 0.1 ml of 50% trichloroacetic acid, and centrifuged at 0°C. Portions (0.15 ml) of the supernatants were diluted with 0.85 ml of water and counted in 10 ml of 2:1 tolueneTriton X-1 00 scintillation fluid containing 0.6% PPO (2,5-diphenyloxazole). Portions (0.25 ml) of neutralized supernatants diluted with water were applied to AG-I-formate anion-exchange columns, and the material not binding to the columns was collected in scintillation vials. (A) Amount ofacid-soluble radioactivity produced in the absence of chloramphenicol or actinomycin D (0) and when actinomycin D is added 2 min before phage (A). The neutral and positively charged compounds produced in the presence of actinomycin D are also shown (A). (B) Amount of acid-soluble radioactivity produced when chloramphenicol is added 2 min before (U) and 6 min after (@). The neutral and positively charged compounds produced when chloramphenicol is added 2 min before phage are also shown (0). All results are expressed as counts per minute per milliliter oforiginal culture. The dashed lines indicate the amount of total radioactivity present in the culture.
? 0.5
of P
N
Q
m~~~~~~~~0.
adatin
c
4
Colun (1C. STANDARDS I 4o
1.0
H,20 URACIL FRAtION NMIE
dUMP 3. eluting solvent (A and B). Uracil and dUUP(0.5 x
C
10
20 30 FRACTION NUMBER
40
30
FIG. 2. Chromatography of acid-soluble products of PBS2 DNA degradation on Rio-Gel P-2. The 15mmn neutralized samples were chromatographed on columns (1.5 by 27 cm) of P-2, using water as the eluting solvent (A and B). Uracil and dUMP (0.5 p.mol of each) were added to each sample as standards, and the positions of their elution are indicated
by the arrows on the elution profiles. The identification ofthese peaks was confirmed by determining the UV spectra of the peak fractions in both acidic and alkaline solution; the peaks were missing when uracil and dUMP were not added to the samples. Uracil and dUMP were also chromatographed with 1 ,uCi of 3H20 obtained from New England Nuclear Corp. (C). The dUMP elutes somewhat earlier on this column than on the sample columns, presumably because of the high concentration of salt in the samples. Two-milliliter fractions were collected.
NOTES
VOL. 22, 1977
cause it adsorbs to the gel filtration matrix. The identity of the radioactivity in fractions 32 to 37 was confirmed by thin-layer chromatography with authentic uracil. Similar results were obtained with the 30-min samples, and the results from the 15- and 30-min samples are summarized in Table 1. The scheme shown in Fig. 3 indicates how PBS2 DNA labeled in the pyrimidine bases may be degraded to yield 3H20 as the major radioactive product. The degradation is pre-
sumably initiated by uracil-DNA N-glycosidase, releasing some of the DNA uracil as the free base. The uracil release probably continues as long as the DNA is a suitable substrate. Each release of uracil creates a substrate site for "apurinic site" endonucleases such as endonuclease II (8), and the major breakdown of the DNA is presumably due to the action of endoand exonucleases subsequent to the action of the uracil-DNA N-glycosidase. Eventually, [53H]dCMP and [5-3H]dUMP are produced, and [5-3H]dCMP can be deaminated to [5-3H]dUMP since B. subtilis contains dCMP deaminase activity (unpublished observation). Water is the ultimate radioactive product since the 3H on the 5-position of dUMP is released during the synthesis of dTMP from dUMP. TABLE 1. Products of in vivo degradation of PBS2 DNA Radioactive acid-soluble products (%)
Oligonucleotides and nu-
Sample
Uracil
Water
5
9 10
75 85
26 23
12 12
62 65
cleotides
Chloramphenicol 16
15 min 30 min
Actinomycin D 15 min 30 min
*
DNA
uracil-
DNA
_rl-il-c
N-gylwuslumw
* * partially DEPYRIMIDINATED DNA + URACIL
nucleuses
dTMP
*
thymidylate
synthetase
dUMP*
dCMP
deominose
CMP*
H20 FIG. 3. Hypothetical pathway for degradation of PBS2 DNA in B. subtilis SB19. Asterisk denotes radioactive compounds.
837
The release of uracil appears to be similar in the presence of chloramphenicol and actinomycin D, but nucleolytic degradation is slower when actinomycin D is present, suggesting that the actinomycin D bound to DNA inhibits this degradation. It is also possible that some uracil is reincorporated into ribonucleotides by uridine monophosphate pyrophosphorylase, and that some of the radioactivity in fractions 12 to 20 is in uridine ribonucleotides. If so, some of this radioactivity should have been incorporated into RNA in the presence of chloramphenicol but not actinomycin D. This does not seem to be the case, as acid solubilization in the presence of chloramphenicol was as rapid and complete as it was in the presence of actinomycin D. The data presented here indicate that parental PBS2 DNA is stable during the normal infection of B. subtilis. Even when phage replication is inhibited by chloramphenicol, the parental PBS2 DNA is not degraded if the chloramphenicol is added after the synthesis of the phage-induced N-glycosidase inhibitor has occurred, suggesting that inhibition of the Nglycosidase is sufficient to prevent degradation of the phage DNA. The parental PBS2 DNA is not subject to attack by the host N-glycosidase for the first 6 min after infection in the presence of chloramphenicol or actinomycin D, but by 10 min this protection or immunity is lost. This protection does not require either protein or RNA synthesis, but the degradation begins at about the same time as the induction of phageinduced enzymes required for the synthesis of progeny phage DNA (5). Perhaps degradation is initiated during conversion of the parental DNA to a state competent for transcription of these enzymes. One possibility is that both this transcription and attack by the N-glycosidase require the generation of single-stranded regions in the parental DNA. Interrupted transfer of the parental DNA into the cell, similar to that observed for some coliphages (6), could also explain the 6-min lag before degradation begins. If interrupted transfer of PBS2 does occur, complete transfer obviously does not require protein synthesis. Whether or not the uracil-DNA N-glycosidase is the enzyme responsible for initiating the rapid degradation of the parental PBS2 DNA has not been unequivocally demonstrated by these experiments. Isolation of a phage mutant unable to induce the N-glycosidase inhibitor would be useful for resolution of this uncertainty. Alternatively, if the experiments described here could be performed in a B. subtilis mutant deficient in the N-glycosidase activity, the parental PBS2 DNA should be more stable.
838
J. VIROL.
NOTES
Such mutants have been isolated in E. coli (B. K. Duncan, P. A. Rockstroh, and H. R. Warner, Fed. Proc. 35:1493, 1976), and the role of the N-glycosidase in excluding uracil from DNA in E. coli cells is currently being studied. The results presented here do indicate that the bacterial N-glycosidase will attack uracil-containing PBS2 DNA if given the chance, and that PBS2 must inhibit this enzyme to survive. In a bacterial cell where the occurrence of uracil in the DNA may be a rare event through misincorporation or deamination of cytosine, it seems reasonable that the N-glycosidase initiates repair of the chromosome rather than massive degradation. This investigation was supported by Public Health Service research grant GM 21464, to H. R. W., and postdoctoral fellowship GM 05401 to B. K. D., both from the National Institute of General Medical Sciences. We thank Alan Price for help and advice in preparing the labeled PBS2.
4.
5.
6. 7.
8. 9. 10.
11. 12.
LITERATURE CITED 1. Bessman, M. J., I. R. Lehman, J. Adler, S. B. Zimmerman, E. S. Simms, and A. Kornberg. 1958. Enzymatic synthesis of deoxyribonucleic acid. III. The incorporation of pyrimidine and purine analogues into deoxyribonucleic acid. Proc. Natl. Acad. Sci. U.S.A. 44:633640. 2. Drake, J. W., and R. H. Baltz. 1976. The biochemistry of mutagenesis. Annu. Rev. Biochem. 45:11-37. 3. Duncan, J., L. Hamilton, and E. Friedberg. 1976. Enzymatic degradation of uracil-containing DNA. II. Evidence for N-glycosidase and nuclease activities in
13.
14.
unfractionated extracts of Bacillus subtilis. J. Virol. 19:338-345. Friedberg, E. C., A. K. Ganesan, and K. Minton. 1975. N-glycosidase activity in extracts of Bacillus subtilis and its inhibition after infection with bacteriophage PBS2. J. Virol. 16:315-321. Katz, G. E., A. R. Price, and M. J. Pomerantz. 1976. Bacteriophage PBS2-induced inhibition of uracil-containing DNA degradation. J. Virol. 20:535-538. Lanni, Y. T. 1968. First-step-transfer deoxyribonucleic acid of bacteriophage T5. Bacteriol. Rev. 32:227-242. Lindahl, T. 1974. An N-glycosidase from Escherichia coli that releases free uracil from DNA containing deaminated cytosine residues. Proc. Natl. Acad. Sci. U.S.A. 71:3649-3653. Lindahl, T. 1976. New class of enzymes acting on damaged DNA. Nature (London) 259:64-66. Price, A. R., and S. J. Cook. 1972. New deoxyribonucleic acid polymerase induced by Bacillus subtilis bacteriophage PBS2. J. Virol. 9:602-610. Price, A. R., and S. M. Fogt. 1973. Deoxythymidylate phosphohydrolase induced by bacteriophage PBS2 during infection of Bacillus subtilis. J. Biol. Chem. 248:1372-1380. Price, A. R., and J. Frato. 1975. Bacillus subtilis deoxyuridine-triphosphatase and its bacteriophage PBS2induced inhibitor. J. Biol. Chem. 250:8804-8811. Takahashi, I., and J. Marmur. 1963. Replacement of thymidylic acid by deoxyuridylic acid in the deoxyribonucleic acid of a transducing phage for Bacillus subtilis. Nature (London) 197:794-795. Tye, B., P. Nyman, I. R. Lehman, S. Hochhauser, and B. Weiss. 1977. Transient accumulation of Okazaki fragments as a result of uracil incorporation into nascent DNA. Proc. Natl. Acad. Sci. U.S.A. 74:154157. Warner, H. R., R. F. Drong, and S. M. Berget. 1975. Early events after infection of Escherichia coli by bacteriophage T5. I. Induction of a 5'-nucleotidase activity and excretion of free bases. J. Virol. 15:273280.
JOURNAL OF VIROLOGY, June 1977, p. 835-838 Copyright © 1977 American Society for Microbiology
Metabolism of Uracil-Containing DNA: Degradation of Bacteriophage PBS2 DNA in Bacillus subtilis1 BRUCE K. DUNCAN AND HUBER R. WARNER* Department of Biochemistry, College of Biological Sciences, University of Minnesota, St. Paul, Minnesota 55108
Received for publication 28 December 1976
When Bacillus subtilis is infected by the uracil-containing DNA phage PBS2, the parental DNA labeled with radioactive uracil and cytosine remains acid insoluble. If the synthesis of the phage-induced uracil-DNA N-glycosidase inhibitor is prevented, the parental DNA is completely degraded to acid-soluble products beginning at about 6 min after infection. The host N-glycosidase probably initiates the degradation pathway, with nucleases being responsible for the remaining degradation of the DNA.
Although the DNA in almost all biological systems contains thymine, a consideration of the known pathways necessary for DNA synthesis suggests that the occasional incorporation of uracil in place of thymine in DNA is at least a possibility (13). Bessman et al. have shown that dUTP is a substrate for Escherichia coli DNA polymerase I in vitro (1), and presumably dUTP could be used by other DNA polymerases as well. In addition, the spontaneous or chemically induced deamination of cytosine in situ would also result in the presence of uracil in DNA and occurs at biologically significant rates (2). The presence of adenine-uracil base pairs in DNA could conceivably cause defective DNA function, whereas the deamination of cytosine to uracil would induce mutations. An enzyme that could play an obvious role in repair of such uracil-containing DNA regions has recently been discovered in E. coli (7) and Bacillus subtilis (4). This enzyme is a uracil-DNA N-glycosidase, which catalyzes the following reaction: DNA -- uracil + apyrimidinic DNA. Thus, uracil can be removed from DNA, and the DNA can be repaired by the sequential action of endonuclease II, DNA polymerase I, and DNA ligase (8). The B. subtilis phage PBS2 is a virus whose DNA contains uracil completely substituted for thymine (12). Several proteins are induced by this phage to facilitate the synthesis of uracilcontaining DNA in cells normally engaged in the synthesis of thymine-containing DNA (3-5, 9-11). One of these proteins is an inhibitor of the B. subtilis uracil-DNA N-glycosidase. Inhibition of this enzyme commences within 1 min 1 Scientific Paper no. 9764 of the Minnesota Agricultural Experiment Station.
after infection and is complete by about 5 min after infection (3-5). We have used this virus system to study the stability of uracil-containing DNA in bacterial cells. Radioactive PBS2 phage were prepared by adding [5-3H]uracil (39 ,uCi/ml; 11 Ci/mmol) to a B. subtilis SB19 culture in TYG medium (5) 5 min after infection with PBS2 and then shaking the culture at 370C for 100 min. The phage were purified from the lysate by CsCl gradient centrifugation and had a specific activity of 6.1 x 104 cpm/10'0 PFU. Both cytosine and uracil are labeled by [5-3H]uracil in this manner (75% of the radioactivity is in cytosine and 25% is in uracil). These radioactive phage were then used to study the stability of uracil-containing DNA in B. subtilis cells under a variety of conditions. When B. subtilis was infected with radioactive phage at a multiplicity of infection of about 0.3, little acid solubilization of the DNA occurred after infection (Fig. 1A). However, when actinomycin D was added to the culture 2 min prior to infection to prevent induction of phageinduced proteins, including the uracil-DNA Nglycosidase inhibitor, rapid and complete acid solubilization of the phage DNA occurred. A similar addition of chloramphenicol also caused acid solubilization of the DNA (Fig. 1B). When the chloramphenicol was added 6 min after the phage, the N-glycosidase should have already been fully inhibited (4, 5), and no significant acid-solubilization was observed. The acid-soluble products were characterized in two ways. Portions (0.75 ml) of the trichloroacetic acid supernatant fractions were neutralized with 0.225 ml of 1 M Tris base, and 0.25-ml portions of these neutralized samples were diluted with 1 ml of water and chromatographed on columns (0.3 by 5.0 cm) of Bio-Rad AG-1-
835
836
NOTES
J. VIROL.
formate anion-exchange resin. Neutral and positively charged compounds were eluted with water, and nucleoside monophosphates were eluted with 0.2 M ammonium formate, pH 5. The amount of radioactivity eluted in the water wash is shown in Fig. 1. Almost no radioactivity was eluted with the monophosphate fraction (data not shown). These results indicate that neutral and positively charged molecules constitute 65 to 95% of the acid-soluble fraction, and that acid-soluble oligonucleotides constitute most of the remainder. The acid-soluble products were further characterized by chromatography of 0.7-ml portions of the neutralized samples on Bio-Gel P-2 columns. These columns had been previously found to be useful for the separation of dTMP, 100%---
A
ACTINOMYCIN
thymidine, and thymine (14). Elution profiles of the 15-min samples are shown in Fig. 2. Small amounts of radioactive material elute in the positions corresponding to oligonucleotides (fractions 12 to 17), mononucleotides (fractions 18 to 20), and uracil (fractions 32 to 37). The major radioactive product (fractions 28 to 30) did not elute as an identifiable pyrimidine derivative. Lyophilization showed that this product is volatile, suggesting that it is 3H2O. We confirmed that 3H2O does elute in fractions 28 to 30 by chromatographing authentic 3H20 along with dUMP and uracil (Fig. 2C). Under the conditions used here, uracil is retarded bd1.5
B 100%---CHLORAMPHENICOL
15 min
'.5~~~~~~~~~~~. 0~~~~~~~~~~~~~~~. .0~~~~~~~~~~~~~ E > B. ACTINOMYC~~~bI - 15 min ~1.0
i15 X~~~~~
FG 1.0
A. CHLORAMPHENICOL -
*
A
0.-
0.
LL B. ACTINOMYCIN CL
E
-15 min
.0
1 .0STN-D 1.0
~~~~~~~~~~C
.
CONTROL 0
10
20
0 30 20 10 MINUTES AFTER INFECTION
Q5>
30
FIG. 1. Acid solubilization of PBS2 DNA after infection ofB. subtilis. B. subtilis SB19 was grown to an absorbency at 650 nm of 0.7 in TYG medium at 37'C. Actinomycin D or chloramphenicol was added 2 min before the radioactive phage, 6 min after the phage, or not at all. The final concentrations of actinomycin D and chloramphenicol, when added, were 20 and 100 pglml, respectively. At various times, 1ml samples were removed, added to 0.1 ml of 50% trichloroacetic acid, and centrifuged at 0°C. Portions (0.15 ml) of the supernatants were diluted with 0.85 ml of water and counted in 10 ml of 2:1 tolueneTriton X-1 00 scintillation fluid containing 0.6% PPO (2,5-diphenyloxazole). Portions (0.25 ml) of neutralized supernatants diluted with water were applied to AG-I-formate anion-exchange columns, and the material not binding to the columns was collected in scintillation vials. (A) Amount ofacid-soluble radioactivity produced in the absence of chloramphenicol or actinomycin D (0) and when actinomycin D is added 2 min before phage (A). The neutral and positively charged compounds produced in the presence of actinomycin D are also shown (A). (B) Amount of acid-soluble radioactivity produced when chloramphenicol is added 2 min before (U) and 6 min after (@). The neutral and positively charged compounds produced when chloramphenicol is added 2 min before phage are also shown (0). All results are expressed as counts per minute per milliliter oforiginal culture. The dashed lines indicate the amount of total radioactivity present in the culture.
? 0.5
of P
N
Q
m~~~~~~~~0.
adatin
c
4
Colun (1C. STANDARDS I 4o
1.0
H,20 URACIL FRAtION NMIE
dUMP 3. eluting solvent (A and B). Uracil and dUUP(0.5 x
C
10
20 30 FRACTION NUMBER
40
30
FIG. 2. Chromatography of acid-soluble products of PBS2 DNA degradation on Rio-Gel P-2. The 15mmn neutralized samples were chromatographed on columns (1.5 by 27 cm) of P-2, using water as the eluting solvent (A and B). Uracil and dUMP (0.5 p.mol of each) were added to each sample as standards, and the positions of their elution are indicated
by the arrows on the elution profiles. The identification ofthese peaks was confirmed by determining the UV spectra of the peak fractions in both acidic and alkaline solution; the peaks were missing when uracil and dUMP were not added to the samples. Uracil and dUMP were also chromatographed with 1 ,uCi of 3H20 obtained from New England Nuclear Corp. (C). The dUMP elutes somewhat earlier on this column than on the sample columns, presumably because of the high concentration of salt in the samples. Two-milliliter fractions were collected.
NOTES
VOL. 22, 1977
cause it adsorbs to the gel filtration matrix. The identity of the radioactivity in fractions 32 to 37 was confirmed by thin-layer chromatography with authentic uracil. Similar results were obtained with the 30-min samples, and the results from the 15- and 30-min samples are summarized in Table 1. The scheme shown in Fig. 3 indicates how PBS2 DNA labeled in the pyrimidine bases may be degraded to yield 3H20 as the major radioactive product. The degradation is pre-
sumably initiated by uracil-DNA N-glycosidase, releasing some of the DNA uracil as the free base. The uracil release probably continues as long as the DNA is a suitable substrate. Each release of uracil creates a substrate site for "apurinic site" endonucleases such as endonuclease II (8), and the major breakdown of the DNA is presumably due to the action of endoand exonucleases subsequent to the action of the uracil-DNA N-glycosidase. Eventually, [53H]dCMP and [5-3H]dUMP are produced, and [5-3H]dCMP can be deaminated to [5-3H]dUMP since B. subtilis contains dCMP deaminase activity (unpublished observation). Water is the ultimate radioactive product since the 3H on the 5-position of dUMP is released during the synthesis of dTMP from dUMP. TABLE 1. Products of in vivo degradation of PBS2 DNA Radioactive acid-soluble products (%)
Oligonucleotides and nu-
Sample
Uracil
Water
5
9 10
75 85
26 23
12 12
62 65
cleotides
Chloramphenicol 16
15 min 30 min
Actinomycin D 15 min 30 min
*
DNA
uracil-
DNA
_rl-il-c
N-gylwuslumw
* * partially DEPYRIMIDINATED DNA + URACIL
nucleuses
dTMP
*
thymidylate
synthetase
dUMP*
dCMP
deominose
CMP*
H20 FIG. 3. Hypothetical pathway for degradation of PBS2 DNA in B. subtilis SB19. Asterisk denotes radioactive compounds.
837
The release of uracil appears to be similar in the presence of chloramphenicol and actinomycin D, but nucleolytic degradation is slower when actinomycin D is present, suggesting that the actinomycin D bound to DNA inhibits this degradation. It is also possible that some uracil is reincorporated into ribonucleotides by uridine monophosphate pyrophosphorylase, and that some of the radioactivity in fractions 12 to 20 is in uridine ribonucleotides. If so, some of this radioactivity should have been incorporated into RNA in the presence of chloramphenicol but not actinomycin D. This does not seem to be the case, as acid solubilization in the presence of chloramphenicol was as rapid and complete as it was in the presence of actinomycin D. The data presented here indicate that parental PBS2 DNA is stable during the normal infection of B. subtilis. Even when phage replication is inhibited by chloramphenicol, the parental PBS2 DNA is not degraded if the chloramphenicol is added after the synthesis of the phage-induced N-glycosidase inhibitor has occurred, suggesting that inhibition of the Nglycosidase is sufficient to prevent degradation of the phage DNA. The parental PBS2 DNA is not subject to attack by the host N-glycosidase for the first 6 min after infection in the presence of chloramphenicol or actinomycin D, but by 10 min this protection or immunity is lost. This protection does not require either protein or RNA synthesis, but the degradation begins at about the same time as the induction of phageinduced enzymes required for the synthesis of progeny phage DNA (5). Perhaps degradation is initiated during conversion of the parental DNA to a state competent for transcription of these enzymes. One possibility is that both this transcription and attack by the N-glycosidase require the generation of single-stranded regions in the parental DNA. Interrupted transfer of the parental DNA into the cell, similar to that observed for some coliphages (6), could also explain the 6-min lag before degradation begins. If interrupted transfer of PBS2 does occur, complete transfer obviously does not require protein synthesis. Whether or not the uracil-DNA N-glycosidase is the enzyme responsible for initiating the rapid degradation of the parental PBS2 DNA has not been unequivocally demonstrated by these experiments. Isolation of a phage mutant unable to induce the N-glycosidase inhibitor would be useful for resolution of this uncertainty. Alternatively, if the experiments described here could be performed in a B. subtilis mutant deficient in the N-glycosidase activity, the parental PBS2 DNA should be more stable.
838
J. VIROL.
NOTES
Such mutants have been isolated in E. coli (B. K. Duncan, P. A. Rockstroh, and H. R. Warner, Fed. Proc. 35:1493, 1976), and the role of the N-glycosidase in excluding uracil from DNA in E. coli cells is currently being studied. The results presented here do indicate that the bacterial N-glycosidase will attack uracil-containing PBS2 DNA if given the chance, and that PBS2 must inhibit this enzyme to survive. In a bacterial cell where the occurrence of uracil in the DNA may be a rare event through misincorporation or deamination of cytosine, it seems reasonable that the N-glycosidase initiates repair of the chromosome rather than massive degradation. This investigation was supported by Public Health Service research grant GM 21464, to H. R. W., and postdoctoral fellowship GM 05401 to B. K. D., both from the National Institute of General Medical Sciences. We thank Alan Price for help and advice in preparing the labeled PBS2.
4.
5.
6. 7.
8. 9. 10.
11. 12.
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