Vol. 136, No. 3
JOURNAL OF BACTERIOLOGY, Dec. 1978, P. 1205-1207 0021-9193/78/0136-1205$02.00/0 Copyright X) 1978 American Society for Microbiology
Printed in U.S.A.
Differential Effect of Hydroxyurea on the Replication of Plasmid and Chromosomal DNA in Bacillus subtilis A. G. SHIVAKUMAR AND DAVID DUBNAU* Department of Microbiology, The Public Health Research Institute of the City of New York, Inc., New York, New York 10016
Received for publication 11 August 1978
The replication in Bacillus subtilis of the staphylococcal R plasmids pE194, pBD15, pUB110, pSA0501, and pSA2100 has been studied in the presence of hydroxyurea. In all cases, an enrichment for covalently closed circular DNA compared with chromosomal DNA was observed. In this respect, hydroxyurea mimics the effect previously observed with pUB110, using strains carrying the conditional mutation dnaA13. This mutation has been reported to affect ribonucleotide reductase (G. W. Bazill and D. Karamata, Mol. Gen. Genet. 117:19-29, 1972). An explanation for these effects is offered, together with some supporting evidence.
The recent finding that several small Staphylococcus aureus R plasmids can replicate and express antibiotic resistance in Bacillus subtilis (3) has encouraged research on recombinant DNA in this organism (4, 6, 9). Aside from their use for molecular cloning, these plasmids are also useful for studies of plasmid biology. pUB110, a small multicopy kanamycin resistance plasmid, can be amplified in certain DNA temperature-sensitive mutants of B. subtilis at a nonpermissive temperature for chromosomal DNA synthesis (12). This facilitates the isolation of large amounts of pure covalently closed circular (CCC) DNA as well as provides insight into the replication requirements of pUB110. In the amplified state, the synthesis of pUB110specific proteins continues, permitting the identification of these proteins in polyacrylamide gels against a relatively reduced background of host synthesis (Shivakumar and Dubnau, manuscript in preparation). However, amplification of plasmids in DNA temperature-sensitive mutants is not a general method, because some of the S. aureus plasmids are themselves temperature sensitive for replication. For instance, the plasmids pE194, pBD15 (a copy control mutant derivative of pE194), pSA0501, and pSA2100 are temperature sensitive for replication in B. subtilis, and strains carrying them can be cured by growth at 450C (Shivakumar, Contente, Hahn, and Dubnau, unpublished data). An alternative amplification technique was devised, based on the observation that dnaA13, when shifted to 450C in the presence of exogenous deoxyadenosine and thymidine, almost immediately stopped synthesizing chromosomal DNA, while pUBllO DNA synthesis continued
(12). Bazill and Karamata (1) have reported that dnaA is the structural gene for ribonucleotide reductase in B. subtilis, and hydroxyurea (HU) has been found to be an inhibitor of this enzyme in Escherichia coli (13) and in phage T4-infected cultures (14). This report shows that the use of HU in the presence of deoxyadenosine and thymidine mimics the differential effect of the dnaA13 lesion on the synthesis of chromosomal and plasmid DNA. Figure 1 shows the effect of HU on DNA synthesis in B. subtilis. HU at a concentration of 0.2 M inhibits DNA synthesis by 70%. Individual strains of B. subtilis carrying several R plasmids (pUBilO [5, 10], pE194 [5, 8], pBD15 [B. Weisblum, M. Graham, T. Gryczan, and Dubnau, J. Bacteriol., in press], pC194 [3, 5, 8], pSA0501 [5, 7], and pSA2100 [5, 7]) were tested for amplification of CCC DNA in the presence of HU. The cultures were grown at 300C in minimal medium containing 0.5% glucose, 0.1% casein hydrolysate, 50 ,ug of tryptophan per ml, 50 jig of threonine per ml, 250 ,ug of deoxyadenosine per ml, 5 ,ug of thymidine per ml, and 5 ,uCi of [3H]thymidine per ml. When a density of 2 x 10' viable cells per ml was reached, one-half of each culture was withdrawn and chilled, and the cells were harvested by centrifugation. HU (0.2 M) was added to the remaining portions, and incubation was continued for 3 h. These cultures were also harvested, and all of the samples were analyzed on cesium chloride-ethidium bromide density gradients as described (12) (Table 1). In each case an enrichment of CCC over chromosomal DNA occurred during incubation in HU. For pUBllO the extent of this enrichment varied with the temperature
1205
J. BACTERIOL.
NOTES
1206
of incubation. Although the extent of amplification observed under these conditions was less than that obtained with pUB110 by using the DNA temperature-sensitive mutants (12), no further attempt has been made to optimize con15
-l%
10
0
E
Minutes FIG. 1. B. subtilis BD437 (met ile) was grown in minimal medium supplemented with glucose (0.5%), casein hydrolysate (0.1%), and deoxyadenosine (200 ug/ml). To portions of the culture, HU was added to the indicated concentrations, together with 5 uCi of [3HJthymidine per ml, and the incorporation of acidprecipitable radioactivity was followed. Symbols: 0, no HU; *, 0.025 M HU; x, 0.05 M HU; U, 0.075 M HU; EL, 0.IOMHU; A, 0.20MHU.
ditions. The electrophoretic mobility on agarose gels of CCC DNA from all the plasmids, isolated after 3 h of incubation with 0.2 M HU, was indistinguishable from that of authentic plasmid DNA (data not shown). The specific transforming activities were also similar with HU-amplified and nonamplified CCC DNA (data not shown). Bazill and Karamata (1) have reported that deoxyadenosine and thymidine could not repair either the dnaA13 lesion or the inhibitory effect of HU on colony formation. However, in the presence of deoxyadenosine and thymidine, pUB110 DNA replication is unaffected in dnaA13 at a temperature that completely inhibits chromosome replication (12). To explain this, we propose that ribonucleotide reductase provides two functions, both of which are essential for chromosomal DNA synthesis. One function is the reduction of ribonucleotides. We suggest that deoxyribonucleotides can also be provided by the addition of exogenous deoxyribonucleosides. The second postulated function is required for the synthesis of chromosomal but not of plasmid DNA. The present results suggest that the effect of HU mimics that of the dnaA13 lesion and results in a defect in both postulated functions. The addition of deoxyadenosine and thymidine, therefore, allows only plasmid DNA synthesis to proceed. As a first step toward testing this hypothesis, we examined the effect of HU on DNA synthesis in toluene-treated cells (2, 11) (Fig. 2). HU (0.2 M) inhibited DNA synthesis by about 50%. Because an excess of all four deoxyribonucleotide triphosphates was present in the toluene-treated cell system, this result is consistent with an additional role for ribonucleotide reductase beside precursor synthesis. Attempts to demonstrate temperature-
TABLE 1. Effect of HU on replication of chromosomal and plasmid DNA
Plasmid
Growth temp
cpm after growth in HU/cpm before HU addition
(00)
CCC DNA
Charomo-NA
sonu DNA
Before HU addition
After growth in HU
Fraction of FatoofCpnucpm in CCC hera beDNA
cpm in CCC DNA
Copy numher0
br
125 0.01 35 0.02 1.9 7.3 81 0.10 40 0.05 1.8 4.0 154 0.19 40 0.05 1.5 6.6 260 0.31 40 0.05 1.3 12.1 46 0.05 8 0.01 11.4 1.8 pSA0501 33 0.06 14 0.03 2.0 5.0 pSA2100 50 0.05 8 0.01 1.8 10.3 pE194 141 0.135 67 0.06 2.0 4.6 pBD15 'The molecular weights used to calculate copy numbers were as follows: chromosome, 2.5 x 10"; pC194, 2.0 x 106; pUBl10, 3.0 x 106; pSA0501, 2.8 x 106; pSA2100, 4.7 x 106; pE194 and pBD15, 2.4 x 106 (5; Weisblum et al., in press).
pC194 pUB110 pUB110 pUB110
37 32 37 45 32 32 32 32
NOTES
VOL. 136, 1978
1207
ature sensitive in the wild type (data not shown). Whatever the explanation for the relative HU resistance of plasmid replication, this system provides a simple method for the amplification of several R plasmids in B. subtilis. It will be of interest to extend these observations to other systems. We express our appreciation to N. Brown for discussion and to A. Howard for expert secretarial assistance. This work was supported by Public Health Service grant AI-10311 from the National Institute of Allergy and Infectious Diseases awarded to D.D.
ft) 0
E
LITERATURE CITED
O
N
0.A
0
10
20
30
Minutes
1. Bazill, G. W., and D. Karamata. 1972. Temperaturesensitive mutants of B. subtilis defective in deoxyribonucleotide synthesis. Mol. Gen. Genet. 117:19-29. 2. Brown, N. C., C. L. Wisseman HI, and T. Matsushita. 1972. Inhibition of bacterial DNA replication by 6-(phydroxyphenylazo)-uracil. Nature (London) New Biol. 237:72-74. 3. Ehrlich, S. D. 1977. Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 74:1680-1682. 4. Ehrlich, S. D. 1978. DNA cloning in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 75:1433-1436. 5. Gryczan, T. J., S. Contente, and D. Dubnau. 1978. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J. Bacteriol. 134:318-329. 6. Gryczan, T. J., and D. Dubnau. 1978. Construction and properties of chimeric plasmids in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 75:1428-1432. 7. Iordanescu, S. 1975. Recombinant plasmid obtained from two different, compatible staphylococcal plasmids. J. Bacteriol. 124:597-601. 8. Iordinescu, S. 1976. Three distinct plasmids originatin g in the same Staphylococcus aureus strain. Arch. Roum. Pathol. Exp. Microbiol. 35:111-118. 9. Keggins, K. M., P. S. Lovett, and E. J. Duvall. 1978. Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB110. Proc. Natl. Acad. Sci. U.S.A.
FIG. 2. Effect ofhydroxyurea on the incorporation of [3H]TTP into DNA of toluene-treated B. subtilis. B. subtilis strain BD274 (trpC2 thr spolA58) was grown in 50 ml of minimal medium at 30°C, to a density of 8 x 167 cells per ml. Cells were collected by filtration, washed in 0.1 M potassium phosphate buffer (pH 7.4), resuspended in 1 ml of phosphate buffer containing 1% toluene, and agitated gently at 25°C for 10 min. The suspension was then incubated for 5 min at 4°C and used immediately. The complete reaction mixture (250 1I) contained 70 mM K2HPO4 (pH 7.4); 13 mM MgSO4; 1.3 mMATP; 33 each of GTP, dCTP, dATP, dTTP, and [3H]dTTP (0.15 ,ul/nmol); 2 mM dithiothreitol; and 2 x 10' toluenetreated cells. Samples (50 ,ul) were removed at inter75:1423-1427. vals to 1 ml of cold 5% trichloroacetic acid. Acidinsoluble material was collected and washed with 10. Lacey, R. W., and I. Chopra. 1974. Genetic studies of a multi-resistant strain of Staphylococcus auretls. J. Med. cold 5% trichloroacetic acid on membrane filters. Microbiol. 7:285-297. Symbols: 0, complete; 0, 0.1 M, hydroxyurea; x, 0.2 11. Matsushita, T., K. P. White, and N. Sueoka. 1971. M hydroxyurea; A, minus dGTP; 4 300 XLAM hydroxChromosome replication in toluenized Bacillus subtilis yphenylazouracil. The dithiothreitol present in the cells. Nature (London) New Biol. 232:111-114. reaction mixture was sufficient to reduce the hydrox- 12. Shivakumar, A. G., and D. Dubnau. 1978. Plasmid replication in dna Ts mutants of Bacillus subtilis. yphenylazouracil to the active derivative (2). In all Plasmid 1:405-416. cases, parallel mixtures lacking ATP were tested. 13. Sinha, N. K., and D. P. Snustad. 1972. Mechanism of These all gave indistinguishable results (E). inhibition of deoxyribonucleic acid synthesis in Esche-
sensitive incorporation into toluene-treated cells of the dnaA13 strain failed, because incorporation in toluene-treated cells is markedly temper-
richia coli by hydroxyurea. J. Bacteriol. 112:1321-1334. 14. Yeh, Y.-C., and L. Te8sman. 1978. Differential effect of hydroxyurea on a ribonucleotide reductase system. J. Biol. Chem. 253:1323-1324.
JOURNAL OF BACTERIOLOGY, Dec. 1978, P. 1205-1207 0021-9193/78/0136-1205$02.00/0 Copyright X) 1978 American Society for Microbiology
Printed in U.S.A.
Differential Effect of Hydroxyurea on the Replication of Plasmid and Chromosomal DNA in Bacillus subtilis A. G. SHIVAKUMAR AND DAVID DUBNAU* Department of Microbiology, The Public Health Research Institute of the City of New York, Inc., New York, New York 10016
Received for publication 11 August 1978
The replication in Bacillus subtilis of the staphylococcal R plasmids pE194, pBD15, pUB110, pSA0501, and pSA2100 has been studied in the presence of hydroxyurea. In all cases, an enrichment for covalently closed circular DNA compared with chromosomal DNA was observed. In this respect, hydroxyurea mimics the effect previously observed with pUB110, using strains carrying the conditional mutation dnaA13. This mutation has been reported to affect ribonucleotide reductase (G. W. Bazill and D. Karamata, Mol. Gen. Genet. 117:19-29, 1972). An explanation for these effects is offered, together with some supporting evidence.
The recent finding that several small Staphylococcus aureus R plasmids can replicate and express antibiotic resistance in Bacillus subtilis (3) has encouraged research on recombinant DNA in this organism (4, 6, 9). Aside from their use for molecular cloning, these plasmids are also useful for studies of plasmid biology. pUB110, a small multicopy kanamycin resistance plasmid, can be amplified in certain DNA temperature-sensitive mutants of B. subtilis at a nonpermissive temperature for chromosomal DNA synthesis (12). This facilitates the isolation of large amounts of pure covalently closed circular (CCC) DNA as well as provides insight into the replication requirements of pUB110. In the amplified state, the synthesis of pUB110specific proteins continues, permitting the identification of these proteins in polyacrylamide gels against a relatively reduced background of host synthesis (Shivakumar and Dubnau, manuscript in preparation). However, amplification of plasmids in DNA temperature-sensitive mutants is not a general method, because some of the S. aureus plasmids are themselves temperature sensitive for replication. For instance, the plasmids pE194, pBD15 (a copy control mutant derivative of pE194), pSA0501, and pSA2100 are temperature sensitive for replication in B. subtilis, and strains carrying them can be cured by growth at 450C (Shivakumar, Contente, Hahn, and Dubnau, unpublished data). An alternative amplification technique was devised, based on the observation that dnaA13, when shifted to 450C in the presence of exogenous deoxyadenosine and thymidine, almost immediately stopped synthesizing chromosomal DNA, while pUBllO DNA synthesis continued
(12). Bazill and Karamata (1) have reported that dnaA is the structural gene for ribonucleotide reductase in B. subtilis, and hydroxyurea (HU) has been found to be an inhibitor of this enzyme in Escherichia coli (13) and in phage T4-infected cultures (14). This report shows that the use of HU in the presence of deoxyadenosine and thymidine mimics the differential effect of the dnaA13 lesion on the synthesis of chromosomal and plasmid DNA. Figure 1 shows the effect of HU on DNA synthesis in B. subtilis. HU at a concentration of 0.2 M inhibits DNA synthesis by 70%. Individual strains of B. subtilis carrying several R plasmids (pUBilO [5, 10], pE194 [5, 8], pBD15 [B. Weisblum, M. Graham, T. Gryczan, and Dubnau, J. Bacteriol., in press], pC194 [3, 5, 8], pSA0501 [5, 7], and pSA2100 [5, 7]) were tested for amplification of CCC DNA in the presence of HU. The cultures were grown at 300C in minimal medium containing 0.5% glucose, 0.1% casein hydrolysate, 50 ,ug of tryptophan per ml, 50 jig of threonine per ml, 250 ,ug of deoxyadenosine per ml, 5 ,ug of thymidine per ml, and 5 ,uCi of [3H]thymidine per ml. When a density of 2 x 10' viable cells per ml was reached, one-half of each culture was withdrawn and chilled, and the cells were harvested by centrifugation. HU (0.2 M) was added to the remaining portions, and incubation was continued for 3 h. These cultures were also harvested, and all of the samples were analyzed on cesium chloride-ethidium bromide density gradients as described (12) (Table 1). In each case an enrichment of CCC over chromosomal DNA occurred during incubation in HU. For pUBllO the extent of this enrichment varied with the temperature
1205
J. BACTERIOL.
NOTES
1206
of incubation. Although the extent of amplification observed under these conditions was less than that obtained with pUB110 by using the DNA temperature-sensitive mutants (12), no further attempt has been made to optimize con15
-l%
10
0
E
Minutes FIG. 1. B. subtilis BD437 (met ile) was grown in minimal medium supplemented with glucose (0.5%), casein hydrolysate (0.1%), and deoxyadenosine (200 ug/ml). To portions of the culture, HU was added to the indicated concentrations, together with 5 uCi of [3HJthymidine per ml, and the incorporation of acidprecipitable radioactivity was followed. Symbols: 0, no HU; *, 0.025 M HU; x, 0.05 M HU; U, 0.075 M HU; EL, 0.IOMHU; A, 0.20MHU.
ditions. The electrophoretic mobility on agarose gels of CCC DNA from all the plasmids, isolated after 3 h of incubation with 0.2 M HU, was indistinguishable from that of authentic plasmid DNA (data not shown). The specific transforming activities were also similar with HU-amplified and nonamplified CCC DNA (data not shown). Bazill and Karamata (1) have reported that deoxyadenosine and thymidine could not repair either the dnaA13 lesion or the inhibitory effect of HU on colony formation. However, in the presence of deoxyadenosine and thymidine, pUB110 DNA replication is unaffected in dnaA13 at a temperature that completely inhibits chromosome replication (12). To explain this, we propose that ribonucleotide reductase provides two functions, both of which are essential for chromosomal DNA synthesis. One function is the reduction of ribonucleotides. We suggest that deoxyribonucleotides can also be provided by the addition of exogenous deoxyribonucleosides. The second postulated function is required for the synthesis of chromosomal but not of plasmid DNA. The present results suggest that the effect of HU mimics that of the dnaA13 lesion and results in a defect in both postulated functions. The addition of deoxyadenosine and thymidine, therefore, allows only plasmid DNA synthesis to proceed. As a first step toward testing this hypothesis, we examined the effect of HU on DNA synthesis in toluene-treated cells (2, 11) (Fig. 2). HU (0.2 M) inhibited DNA synthesis by about 50%. Because an excess of all four deoxyribonucleotide triphosphates was present in the toluene-treated cell system, this result is consistent with an additional role for ribonucleotide reductase beside precursor synthesis. Attempts to demonstrate temperature-
TABLE 1. Effect of HU on replication of chromosomal and plasmid DNA
Plasmid
Growth temp
cpm after growth in HU/cpm before HU addition
(00)
CCC DNA
Charomo-NA
sonu DNA
Before HU addition
After growth in HU
Fraction of FatoofCpnucpm in CCC hera beDNA
cpm in CCC DNA
Copy numher0
br
125 0.01 35 0.02 1.9 7.3 81 0.10 40 0.05 1.8 4.0 154 0.19 40 0.05 1.5 6.6 260 0.31 40 0.05 1.3 12.1 46 0.05 8 0.01 11.4 1.8 pSA0501 33 0.06 14 0.03 2.0 5.0 pSA2100 50 0.05 8 0.01 1.8 10.3 pE194 141 0.135 67 0.06 2.0 4.6 pBD15 'The molecular weights used to calculate copy numbers were as follows: chromosome, 2.5 x 10"; pC194, 2.0 x 106; pUBl10, 3.0 x 106; pSA0501, 2.8 x 106; pSA2100, 4.7 x 106; pE194 and pBD15, 2.4 x 106 (5; Weisblum et al., in press).
pC194 pUB110 pUB110 pUB110
37 32 37 45 32 32 32 32
NOTES
VOL. 136, 1978
1207
ature sensitive in the wild type (data not shown). Whatever the explanation for the relative HU resistance of plasmid replication, this system provides a simple method for the amplification of several R plasmids in B. subtilis. It will be of interest to extend these observations to other systems. We express our appreciation to N. Brown for discussion and to A. Howard for expert secretarial assistance. This work was supported by Public Health Service grant AI-10311 from the National Institute of Allergy and Infectious Diseases awarded to D.D.
ft) 0
E
LITERATURE CITED
O
N
0.A
0
10
20
30
Minutes
1. Bazill, G. W., and D. Karamata. 1972. Temperaturesensitive mutants of B. subtilis defective in deoxyribonucleotide synthesis. Mol. Gen. Genet. 117:19-29. 2. Brown, N. C., C. L. Wisseman HI, and T. Matsushita. 1972. Inhibition of bacterial DNA replication by 6-(phydroxyphenylazo)-uracil. Nature (London) New Biol. 237:72-74. 3. Ehrlich, S. D. 1977. Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 74:1680-1682. 4. Ehrlich, S. D. 1978. DNA cloning in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 75:1433-1436. 5. Gryczan, T. J., S. Contente, and D. Dubnau. 1978. Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J. Bacteriol. 134:318-329. 6. Gryczan, T. J., and D. Dubnau. 1978. Construction and properties of chimeric plasmids in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 75:1428-1432. 7. Iordanescu, S. 1975. Recombinant plasmid obtained from two different, compatible staphylococcal plasmids. J. Bacteriol. 124:597-601. 8. Iordinescu, S. 1976. Three distinct plasmids originatin g in the same Staphylococcus aureus strain. Arch. Roum. Pathol. Exp. Microbiol. 35:111-118. 9. Keggins, K. M., P. S. Lovett, and E. J. Duvall. 1978. Molecular cloning of genetically active fragments of Bacillus DNA in Bacillus subtilis and properties of the vector plasmid pUB110. Proc. Natl. Acad. Sci. U.S.A.
FIG. 2. Effect ofhydroxyurea on the incorporation of [3H]TTP into DNA of toluene-treated B. subtilis. B. subtilis strain BD274 (trpC2 thr spolA58) was grown in 50 ml of minimal medium at 30°C, to a density of 8 x 167 cells per ml. Cells were collected by filtration, washed in 0.1 M potassium phosphate buffer (pH 7.4), resuspended in 1 ml of phosphate buffer containing 1% toluene, and agitated gently at 25°C for 10 min. The suspension was then incubated for 5 min at 4°C and used immediately. The complete reaction mixture (250 1I) contained 70 mM K2HPO4 (pH 7.4); 13 mM MgSO4; 1.3 mMATP; 33 each of GTP, dCTP, dATP, dTTP, and [3H]dTTP (0.15 ,ul/nmol); 2 mM dithiothreitol; and 2 x 10' toluenetreated cells. Samples (50 ,ul) were removed at inter75:1423-1427. vals to 1 ml of cold 5% trichloroacetic acid. Acidinsoluble material was collected and washed with 10. Lacey, R. W., and I. Chopra. 1974. Genetic studies of a multi-resistant strain of Staphylococcus auretls. J. Med. cold 5% trichloroacetic acid on membrane filters. Microbiol. 7:285-297. Symbols: 0, complete; 0, 0.1 M, hydroxyurea; x, 0.2 11. Matsushita, T., K. P. White, and N. Sueoka. 1971. M hydroxyurea; A, minus dGTP; 4 300 XLAM hydroxChromosome replication in toluenized Bacillus subtilis yphenylazouracil. The dithiothreitol present in the cells. Nature (London) New Biol. 232:111-114. reaction mixture was sufficient to reduce the hydrox- 12. Shivakumar, A. G., and D. Dubnau. 1978. Plasmid replication in dna Ts mutants of Bacillus subtilis. yphenylazouracil to the active derivative (2). In all Plasmid 1:405-416. cases, parallel mixtures lacking ATP were tested. 13. Sinha, N. K., and D. P. Snustad. 1972. Mechanism of These all gave indistinguishable results (E). inhibition of deoxyribonucleic acid synthesis in Esche-
sensitive incorporation into toluene-treated cells of the dnaA13 strain failed, because incorporation in toluene-treated cells is markedly temper-
richia coli by hydroxyurea. J. Bacteriol. 112:1321-1334. 14. Yeh, Y.-C., and L. Te8sman. 1978. Differential effect of hydroxyurea on a ribonucleotide reductase system. J. Biol. Chem. 253:1323-1324.
