JOURNAL OF BACTERIOLOGY, Feb. 1975, p. 537-547 Copyright 0 1975 American Society for Microbiology
Vol. 121, No. 2 Printed in U.SA.
Sedimentation Analysis of Deoxyribonucleic Acid from Thymine-Starved Escherichia coli HIROAKI NAKAYAMA' AND PHILIP HANAWALT* Department of Biological Sciences, Stanford University, Stanford, California 94305 Received for publication 8 November 1974
During thymine starvation, strand breaks accumulate in the chromosomal deoxyribonucleic acid (DNA) of Escherichia coli. This effect occurs to a varying extent in different strains and is particularly enhanced in strains deficient in DNA polymerase I. The inhibition of ribonucleic acid or protein synthesis suppresses the accumulation of strand breaks. In a polA strain, rifampin is more effective than chloramphenicol or puromycin in suppressing strand break accumulation. To a certain extent the phenomenon of thymineless death correlates with the appearance of strand breaks. Although the killing can not be explained by the bulk of strand breaks, it is possible that some of them represent lethal events. On the basis of our observations we proposed the following model. (i) Transcription may be accompanied by single-strand breaks in DNA. (ii) DNA polymerase I is involved in the efficient repair of these breaks. (iii) Thymine deprivation results in the accumulation of unrepaired breaks. (iv) Polymerase I-mediated repair is less affected by thymine deprivation than are the alternative pathways because it closes the breaks with short patches, requiring less thymine. When growing cells of thymine-requiring Escherichia coli are transferred to a medium that lacks thymine but is otherwise sufficient for continued growth, loss of viability (i.e., thymineless death) occurs (7). Despite numerous studies, the precise mechanism for this phenomenon is unknown. Models that have been advanced to explain thymineless death include unbalanced growth (6, 7), prophage induction (24, 28), deoxyribonucleic acid (DNA) transcription (18, 20), and DNA damage (29), including possible damage due to transcription (35). * Since thymine starvation is known to be mutagenic (9, 25, 44) and recombinogenic (17) and to result in repair replication (35), models involving DNA damage as a proximal cause of death have been attractive and provoked an extensive search for such damage. Menningmann and Szybalski (29) presented evidence that single-strand breaks occur in the DNA of thymine-deprived Bacillus subtilis cells. More recently, Friefelder (14) definitively showed that single-stranded breaks occur in a circular plasmid DNA during thymine starvation of its thymine-requiring E. coli host. As for the chromosomal DNA of E. coli, however, there have appeared several conflicting reports. Walker (42) and Reichenbach et al. (36) detected strand breaks, whereas others (2, 38) found no effect of Present address: Department of Biochemistry, Kyushu Dental College, Kokura, Kitakyushu, Japan. 537 I
thymine deprivation on the integrity of chromosomal DNA. However, different strains of E. coli were used in each of these studies. In the present study, we have attempted to answer the following questions. (i) Does thymine starvation induce DNA breaks in all E. coli strains, or is the effect strain dependent? (Since alkaline sucrose gradient centrifugation cannot discriminate single- and double-strand breaks and alkali-labile linkages, reference to "strand breaks" in this paper always implies the possibility of any or all of these structural defects.) (ii) What is the mechanism for producing the strand breaks? (iii) Are they causally related to thymineless death? Our results indicate that single-strand breaks do appear in the chromosomal DNA during thymine starvation in various E. coli strains, but that the rate of their induction is strain dependent. In addition, an examination of the effect of DNA polymerase I deficiency and various metabolic treatments on the induction of such strand breaks has led us to a working model for the origin of these breaks. We could not, however, reach a definite conclusion as to their relation to thymineless death. (A preliminary account of this work has been presented [N. Nakayama and P. Hanawalt, Fed. Proc. 31:425, 1972].) MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains used are listed in Table 1. Tpis(hy-
538
NAKAYAMA AND HANAWALT
J. BACMEIOL.
TABLE 1. Bacterial strains Strain polA
W3110 thy P3478 PR3478 P112tr PR112tr MM383 MM384 JG138 JG139 C thy-231 HN6
Allele
Other properties
Source and/or reference
F-thy thyR F-thy thyR F-thy thyR F-thy thyR metE F-thy thyR metE F-thy thyR rha lac str F-thy thyR rha lac str F-thy thyR rha lac str F-thy thyR rha lac str E. coli C thy thyR E. coli C thy thyR metE
DeLucia and Cairns (10) DeLucia and Cairns (10) + Cairnsa 3 Cairns' + Cairnsc 12 Monk and Kinross (30) Monk and Kinross (30) + 1 Monk et al. (31) + Monk et al. (31) Hewitt et al. (23) + + This work. Mutant from ultravioletmutagenized C thy-321 1 E. coli C thy thyR HN9 This work. P1 transductant of HN6. Donor: JG138. E. coli C thy thyR HN10 This work. P1 transductant of HN6. + Donor: JG138. E. coli B thy thyR ton sul B/r thy Freifelder (13) + a polA+ revertant from P3478 selected on plates containing methyl methane sulfonate. b Temperature-insensitive derivative of strain P112. For derivation of P112, see reference 4. C polA+ revertant from P112tr selected on plates containing methyl methane sulfonate. + 1
droxymethyl)aminomethane (Tris)-salts medium base and Tris minimal medium were as described previously (32). Thymine was added at 2 pg/ml. In some experiments Casamino Acids (Difco, vitamin free) was added to 0.1%. Nutrient agar was 2.3% nutrient agar (Difco) supplemented with 0.25% NaCl. Nutrient soft agar was 0.8% nutrient broth (Difco) containing 0.25% NaCl and solidified with 0.7% agar (Difco). NET buffer consisted of 0.01 M Trishydrochloride, 0.1 M NaCl and 0.1 M ethylenediaminetetraacetate (EDTA), pH 8.1. Unless otherwise specified, cells were grown in flasks at 37 C with reciprocal shaking (ca. 60 cycles/ min). The culture volume was less than 20% of the total capacity of each flask. Change of media was accomplished by harvesting the cells on a Millipore HA filter (0.45-jum pore size). The cells were rinsed with Tris-salts medium base and suspended in the desired medium. Assay of viable cell concentration was accomplished by appropriate dilution of samples in Trissalts medium base and plating on nutrient agar plates using the soft-agar overlay method (3 ml of soft agar per plate). Radioistopic labeling of cellular DNA. An overnight culture was diluted 20- to 40-fold into a small volume (usually 1 or 2 ml) of radioisotopic labeling medium, and cells were allowed to grow to a cell density of 3 x 108 to 5 x 108/ml. The culture was diluted 10-fold with nonradioactive medium and allowed to grow for about one generation. The resulting mid-log-phase cells were then transferred to the desired medium for each experiment. The labeling medium was Tris-minimal supplemented with either [14Clthymine (1 juCi/ml, 2.1 lAg/ml) or ['HIthymine (20 pCi/ml, 2.1 ug/ml). Sedimentation of DNA through alkaline sucrose gradIents. A culture to be analyzed was chilled in an ice-water bath, and cells were collected by centrifugation at 7,000 x g at 4 C for 3 min in a Sorval RC2-B
centrifuge, washed once with chilled NET buffer, and finally suspended in chilled 15% (wt/vol) sucrose in NET buffer at a density of 10' to 1.5 x 10'/ml. (For comparison of two separate cultures labeled with '4C and 'H, respectively, they were mixed after being chilled prior to Sorval centrifugation.) A 0.1 volume of lysozyme solution (Boehringer, 20 mg/ml in NET buffer) was added, and the mixture was kept in an ice-water bath for 15 min. A 0.02-ml volume of the resulting spheroplast suspension (containing 2 x 107 to 3 x 107 spheroplasts) was then gently mixed into 0.1 ml of a lysing solution (15% [wt/volI sucrose-i M NaOH-0.01 M EDTA) layered on top of a 4.85-ml linear alkaline sucrose gradient (20 to 33% [wt/voll sucrose-0.5 M NaOH-0.5 M NaCl-0.005 M EDTA). After standing at room temperature for 15 to 30 min, gradients were centrifuged at 36,000 rpm at 20 C for 120 min in an SW39L rotor. About 32 fractions were collected dropwise from the bottom of the tube, and the acid-insoluble radioactivity in each fraction was assayed as described by Hanawalt and Cooper (21). This type of sucrose gradient was calibrated by using phage T6 DNA labeled with 140C]thymine, and confirmed to be practically isokinetic. Number average molecular weight (Mn) of DNA in the gradient was calculated with the equation: Mn=
if,
Z(fJMj
where f, is the percentage of radioactivity in fraction i, and M, is the molecular weight of a homogeneous DNA that would have its maximal concentration in fraction i. For the sake of reproducibility, fractions 9 through 28 were used for the calculation of average molecular weight. M, was obtained by the equation (1):
D, D2
(M) 0.38 \M2/
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539
which relates the molecular weights of two DNA preparations (M1 and M2) to the distance sedimented (D, and D,). "4C-labeled T6 DNA was used as a reference. Chemicals. [3H]thymine and [14C]thymine were purchased from New England Nuclear. Chloramphenicol, rifampin, and 5-methyltryptophan were from Calbiochem. Puromycin was obtained from Nutritional Biochemicals Corp.
RESULTS
Generality of thymineless-induced strand breaks and effect of polA mutation. In view of the conflicting reports as to the occurrence of thymineless-induced strand breaks in the chromosomal DNA of E. coli, we first examined sedimentation patterns of DNA from thyminestarved cells of various E. coli strains. Particular emphasis was placed on polA- strains, since one such mutant, P3478 polAl, has been reported to be unusually sensitive to thymineless death (38, 40; H. E. Bendigkeit and P. C. Hanawalt, Bacteriol. Proc., p. 36, 1968). Recently, Berg and O'Neill (5) reported similar results with polA6 as well as polAl mutants. Figure 1 shows typical results for strains W3110 thy- and P3478. In strain W3110 thy-, there was a very slow but progressive change in the DNA sedimentation pattern during a 180min period of thymine starvation. Thus, the peak became broader with time, and the position of the peak fraction shifted considerably toward the top of the gradient. Despite the apparent change in sedimentation profile, the average molecular weight of single-strand DNA decreased only by a factor of 1.6. In contrast, in strain P3478 the change in the shape of the sedimentation pattem occurred earlier (already apparent at 60 min), and the number of strand breaks for all time points examined was greater than that in strain W3110 thy-. After 180 min of thymine starvation, the reduction of average molecular weight was by a factor of 2.2. Strain PR3478, a poUA+ revertant from strain P3478, was indistinguishable from strain W3110 thyin this type of experiment (data not shown). This effect of the polAl allele was also confirmed by using a transductant pair, JG138 and JG139, although the accumulation of strand breaks was much less pronounced in strain JG139 polA+ than in strain W3110 thy-. In spite of the fact that these were apparently the same strains used by Sedgwick and Bridges (38), we have not been able to confirm their inability to find strand breaks induced by thymineless incubation. To ascertain whether the enhanced accumulation of strand breaks in polAl strains was actually the consequence of DNA polymerase I
'. 7.
cX 10 _
5
,, 1 120
00
I
.0~~~~~~~
10 12 3mi 0~~~~~~~. 50~~~~~~~~~~~~~~~~~1
Q 00
10
20 Froction No.
30
FIG. 1. Alkaline sucrose gradient sedimentation profiles of DNA from thymine-starved and nonstarved cells of strains W3110 thy- and P3478. Cells were labeled with [3H]thymine (strain W3110) or ["4C]thymine (strain P3478) and starved for thymine for the times indicated. The control (0 min) consisted of cells suspended in thymineless medium but kept in an ice-water bath until analyzed. The two cultures were then mixed and analyzed. Symbols: 0, strain W3110 thy-; *, strain P3478. Number average molecular weight for each profile is: strain W3110 thy-, 1.4 x 10' (0 min), 1.2 x 10' (60 min), 1.1 x 108 (120 min), and 8.9 x 107 (180 min); strain P3478, 1.3 x 10' (0 min), 6.7 x 107 (60 min), 6.3 x 107 (120 min), and 5.9 x 107 (180 min). The peak fraction for the both controls (fraction no. 14) corresponds to 3.4 x 10' molecular weight. Approximate survival level for each starvation period was: strain W3110 thy- 110%o (60 min), 50% (120 min), and 10%o (180 min); strain P3478, 70%o (60 min), 1% (120 min), and 0.2%o (180 min).
deficiency, the effects of two other polA mutations, polA12 and polA3, were examined. Figure 2 illustrates the results obtained with strains MM383 polA12 and MM384 polA+. Since a polA12 mutant produces a thermolabile DNA polymerase I (30), thymine starvation was carried out at both permissive (30 C) and nonpermissive (43 C) temperatures. Strain MM384 exhibited little, if any, change in sedimentation profile at 30 C for up to 150 min, but showed
NAKAYAMA AND HANAWALT
540
J. BACTERIOL.
2 0
0
Z, t a 10 c)
w
Froction No.
FIG. 2. Alkaline sucrose gradient sedimentation profiles of DNA from thymine-starved and nonstarved cells of strains MM383 and MM384. Cultures were labeled with [9H]thymine (strain MM384) or [14C]thymine (strain MM383) and then starved for thymine at 30 or 42 C for the times indicated. The culture media included Casamino Acids. The two cultures were mixed and analyzed. Symbols: 0, strain MM383; 0, strain MM384.
substantial broadening and shift of the peak at 42 C. In strain MM383, on the other hand, a change in sedimentation pattern was evident even at 30 C, and at 42 C, it was much greater than that in strain MM384. The polA3 mutation also exhibited a similar enhancing effect on the accumulation of thymineless-induced strand breaks in experiments with strains P112tr and PR112tr (data not shown). Strain PR112tr showed only slight change in sedimentation profile during 240 min of thymine starvation (in the presence of Casamino Acids). The above results were obtained by comparing strains in pairs, each of which was almost isogenic except for the polA locus. Furthermore, three independent polA mutations exerted a similar enhancing effect on the accumulation of thymineless-induced strand breaks. Since polA12 is considered to be a missence mutation, a contribution to the present phenomenon by a possible polar effect of the polAl amber mutation on adjacent genes may be eliminated. Therefore, we conclude that the observed effect on thymineless-induced strand breaks is actually due to the deficiency in DNA polymerase I. Baker and Hewitt (2) reported almost no strand breaks in thymine-starved E. coli C thy-321. We reexamined this same strain to find that it behaved in a unique manner. The DNA sedimentation profile in this strain broadened at an early stage of thymine starvation (30 and 60 min), then returned completely to the original shape (120 min) and remained that way until 240 min (Fig. 3). By 300 min, however, a
small number of strand breaks seemed to have reappeared. This early change in the sedimentation pattern was reproducible, although it had not been seen by Baker and Hewitt (2). The subsequent restoration of the normal profile seemed to be in agreement with their observations. Figure 3 shows that the polAl mutation enhances the accumulation of strand breaks in this strain as well. Strain B/r thy-, a representative of B series strains,' was also tested and found to exhibit about as many strand breaks as strain P3478 under the same conditions (data not shown). Effects of metabolic inhibitors on thymineless-induced strand breaks. The experiments described so far have established that strand breaks accumulate in the chromosomal DNA of thymine-starved cells in the majority of strains tested. The next aspect concerns the mechanism for their formation and the possibility of a causal relationship between strand breaks and thymineless death. The effects of various metabolic manipulations on cells undergoing thymineless death were studied. The results are summarized in Fig. 4, and the following statements may be made within the limits of these experiments. In strain W3110 thy-, the formation of thymineless-induced strand breaks seems to be almost completely blocked by energy deprivation (glucose starvation or cyanide), starvation for inorganic phosphate, inhibition of protein synthesis (chloramphenicol, 5 ,ug/ml or higher; puromycin; and 5-methyltryptophan) or ribonucleic acid synthesis inhibition (rifampin).
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DNA STRAND BREAKS AND THYMINELESS DEATH
In strain P3478 polA -, on the other hand, only deprivation of energy source (glucose starvation) and ribonucleic acid synthesis inhibition (rifampin) were as effective in preventing thymineless-induced strand breaks as in strain W3110 thy-. (Since thymine starvation in the presence of 0.01 M NaCN caused substantial cell lysis in strain P3478 for unknown reasons, the effect of cyanide could not be evaluated.) Starvation for inorganic phosphate and chloramphenicol (even at 25 Ag/ml) exerted only a partial prevention of strand break accumulation, whereas puromycin showed only little, if any, effect in strain P3478. Rejoining of thyminless-induced strand breaks. Thymineless-induced strand breaks can be rejoined when thymine is added back to a starved culture. Figure 5 shows the results of an experiment in which strains P3478 and PR3478 were starved for thymine for varying lengths of time and then allowed to grow in the presence of thymine for 15 min. The DNA was analyzed in alkaline sucrose gradients before and after the 15-min incubation period with thymine. Although each set of thymine starvation periods was primarily chosen so that the two strains give similar survivals, they resulted in comparable sedimentation profiles as well
(i.e., similar numbers of strand breaks) for DNA from both strains. The examination of postincubation sedimentation patterns has revealed obvious rejoining of the strand breaks, as evidenced by restoration of a normal or nearly normal profile, but no evident difference between wild-type and polA strains. This suggests that the polA mutation does not grossly affect the rate of rejoining of the strand breaks after restoration of thymine. It must also be pointed out that, although return to the normal sedimentation profile effected by the 15-min incubation period with thymine was nearly complete after 180 min (for strain PR3478) or 90 min (for strain P3478) of thymine starvation, the survival level under these conditions was only about 10% (see below and Fig. 7 and 8). Survival studies. To clarify the relationship between thymineless-induced strand breaks and thymineless death, the effects upon viability of the polA mutation and the various metabolic treatments were examined. As clearly seen in Fig. 6 each of three polA alleles rendered the cells hypersensitive to thymineless death. It is noted that strain MM383 died faster than strain MM384 even at 30 C, a permissive temperature. This finding, consistent with the analysis of
[_S
0
541
'sI
0
C.) 0
0
.:
KZ
Fraction No.
3. Alkaline sucrose sedimentation profiles of DNA from thymine-starved and nonstarved cells of strains C thy-321 and HN9. Cells were labeled with [3H]thymine (strain C thy-321) or [14C]thymine (strain HN9), thymine-starved for various lengths of time, and then analyzed. The experiment with strain HN9 has been done with 'IC-labeled, thymine-starved or nonstarved cells of strain HN10 as internal controls. Strain-HN10 behaved in exactly the same way as strain C thy-321. Left, Strain C thy-321; right, strain HN9. Symbols: *, nonstarved; 0, starved for 30 min; A, 60 min; A, 120 min; *, 180 min; 0, 240 min; and *, 300 min. FIG.
J. BACTERIOL.
NAKAYAMA AND HANAWALT
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-.1
a 4-
0 .O
0
V0
10
20
30
10
20
30
10
20
30
Fraction No. FIG. 4. Effects of various treatments of DNA sedimentation profiles of thymine-starved cells. Cells of strains W3110 thy- and P3478 were labeled with [3H]thymine and then starved for thymine for 180 min (strain W3110 thy-) or 120 min (strain P3478) with or without various treatments. Each treatment was introduced simultaneously with thymine deprivation and continued throughout the starvation period. Each experiment was monitored by including appropriate internal control (not shown in the figure) labeled with [14Clthymine (nonstarved cells or cells thymine-starved without additional treatment). Symbols: 0, strain P3478; 0, strain W3110 thy-. (a) Nonstarved; (b) thymine starved (- 7T); (c) T, starved for glucose; (d) T, NaCN (0.01 M), (e) T, starved for phosphate; (f) T, chloramphenicol (25 Ag/ml); (g) T, 5-methyltryptophan (200 ug/ml); (h) -T, puromycin (2,000 gg/ml); (i) -T, rifampicin (30 Ag/ml). In experiments with rifampin, cells were pretreated with EDTA according to Rose et at. (37), except that the drug was added immediately after dilution following EDTA treatment. A control experiment showed that this conditioning of the cells did not influence the accumulation of thymineless-induced strand breaks. -
-
-
strand breaks (Fig. 2), suggests that some DNA polymerase I deficiency is manifest in this strain even at the permissive temperature. It is also worthy of note that in strain PR112tr thymine starvation for 240 min, which caused only few strand breaks, resulted in significant killing, survival being about 10%. The effects on thymineless death of various metabolic treatments in strains W3110 thyand P3478 are presented in Fig. 7 and 8. In strain W3110 thy-, cyanide, absence of glucose or inorganic phosphate, chloramphenicol (25
-
-
lsg/ml or higher), puromycin, and rifampin have nearly eliminated thymineless death. Those effects appear to agree with the almost complete inhibition by the same treatments of thymineless-induced strand breaks. In contrast, however, chloramphenicol at a lower concentration (5 Ag/ml) and 5-methyltryptophan are only partially effective in preventing thymineless death in spite of their virtually perfect blocking of the induction of strand breaks. In strain P3478 only glucose starvation resulted in almost complete prevention of thymineless death. Re-
VOL. 121, 1975
DNA STRAND BREAKS AND THYMINELESS DEATH
0o
543
Cwc) l Cd)
10
0 0
10 iW/\
10
20
3 Fraction No.
10
20
30
FIG. 5. Rejoining of thymineless-induced strand breaks. Cells of strains PR3478 and P3478 were labeled with
[3 H)thymine and [14C]thymine, respectively, and then starved for thymine for various times. Thymine was added back to each culture at 2 1gg/ml, and further incubation was made for 15 min. Sampling was carried out
before and after the incubation with thymine. Samples of strains PR3478 and P3478 were combined, as indicated below, soI0that survivals for the two cultures mixed were approximately the same, and analyzed. Symbols: O, strain P3478; *, strain PR3478. (a) Thymine starved for 120 min (PR3478) or 60 min (P3478); (b) thymine starved as in (a) and incubated with thymine for 15 min; (c) thymine starved for 180 min (PR3478) or 90 min (P3478); (d) thymine starved as in (c) and incubated with thymine for 15 min; (e) thymine starved for 250 min (PR3478) or 120 min (P3478); (f) thymine starved as in (e) and incubated with thymine for 15 min. Approximate survivals were 50, 10, and 1% for (a), (c), and (e), respectively.
10-2
0
60 120
180 240 0 6 20 Time of Thymine Storvrtion (min)
180
240
FIG. 6. Thymineless death at various polA8tand polA + strains. Symbols:P, (a) strain W3 10 l, strain thyw; i tr; , strain Pw12 tr. (b) S, strain MM384 ats 30 C; , strain MM383 at 30oC; , strain , strain PR112 MM384 at42bC; , strain MM383 at The culture media used in the experiments involving P112tr 42sC. PR1 12tr, MM383, and MM384 contained Casamino Acids.
P34b78;
544
NAKAYAMA AND HANAWALT
Vol. 121, No. 2 Printed in U.SA.
Sedimentation Analysis of Deoxyribonucleic Acid from Thymine-Starved Escherichia coli HIROAKI NAKAYAMA' AND PHILIP HANAWALT* Department of Biological Sciences, Stanford University, Stanford, California 94305 Received for publication 8 November 1974
During thymine starvation, strand breaks accumulate in the chromosomal deoxyribonucleic acid (DNA) of Escherichia coli. This effect occurs to a varying extent in different strains and is particularly enhanced in strains deficient in DNA polymerase I. The inhibition of ribonucleic acid or protein synthesis suppresses the accumulation of strand breaks. In a polA strain, rifampin is more effective than chloramphenicol or puromycin in suppressing strand break accumulation. To a certain extent the phenomenon of thymineless death correlates with the appearance of strand breaks. Although the killing can not be explained by the bulk of strand breaks, it is possible that some of them represent lethal events. On the basis of our observations we proposed the following model. (i) Transcription may be accompanied by single-strand breaks in DNA. (ii) DNA polymerase I is involved in the efficient repair of these breaks. (iii) Thymine deprivation results in the accumulation of unrepaired breaks. (iv) Polymerase I-mediated repair is less affected by thymine deprivation than are the alternative pathways because it closes the breaks with short patches, requiring less thymine. When growing cells of thymine-requiring Escherichia coli are transferred to a medium that lacks thymine but is otherwise sufficient for continued growth, loss of viability (i.e., thymineless death) occurs (7). Despite numerous studies, the precise mechanism for this phenomenon is unknown. Models that have been advanced to explain thymineless death include unbalanced growth (6, 7), prophage induction (24, 28), deoxyribonucleic acid (DNA) transcription (18, 20), and DNA damage (29), including possible damage due to transcription (35). * Since thymine starvation is known to be mutagenic (9, 25, 44) and recombinogenic (17) and to result in repair replication (35), models involving DNA damage as a proximal cause of death have been attractive and provoked an extensive search for such damage. Menningmann and Szybalski (29) presented evidence that single-strand breaks occur in the DNA of thymine-deprived Bacillus subtilis cells. More recently, Friefelder (14) definitively showed that single-stranded breaks occur in a circular plasmid DNA during thymine starvation of its thymine-requiring E. coli host. As for the chromosomal DNA of E. coli, however, there have appeared several conflicting reports. Walker (42) and Reichenbach et al. (36) detected strand breaks, whereas others (2, 38) found no effect of Present address: Department of Biochemistry, Kyushu Dental College, Kokura, Kitakyushu, Japan. 537 I
thymine deprivation on the integrity of chromosomal DNA. However, different strains of E. coli were used in each of these studies. In the present study, we have attempted to answer the following questions. (i) Does thymine starvation induce DNA breaks in all E. coli strains, or is the effect strain dependent? (Since alkaline sucrose gradient centrifugation cannot discriminate single- and double-strand breaks and alkali-labile linkages, reference to "strand breaks" in this paper always implies the possibility of any or all of these structural defects.) (ii) What is the mechanism for producing the strand breaks? (iii) Are they causally related to thymineless death? Our results indicate that single-strand breaks do appear in the chromosomal DNA during thymine starvation in various E. coli strains, but that the rate of their induction is strain dependent. In addition, an examination of the effect of DNA polymerase I deficiency and various metabolic treatments on the induction of such strand breaks has led us to a working model for the origin of these breaks. We could not, however, reach a definite conclusion as to their relation to thymineless death. (A preliminary account of this work has been presented [N. Nakayama and P. Hanawalt, Fed. Proc. 31:425, 1972].) MATERIALS AND METHODS Bacterial strains and growth conditions. The bacterial strains used are listed in Table 1. Tpis(hy-
538
NAKAYAMA AND HANAWALT
J. BACMEIOL.
TABLE 1. Bacterial strains Strain polA
W3110 thy P3478 PR3478 P112tr PR112tr MM383 MM384 JG138 JG139 C thy-231 HN6
Allele
Other properties
Source and/or reference
F-thy thyR F-thy thyR F-thy thyR F-thy thyR metE F-thy thyR metE F-thy thyR rha lac str F-thy thyR rha lac str F-thy thyR rha lac str F-thy thyR rha lac str E. coli C thy thyR E. coli C thy thyR metE
DeLucia and Cairns (10) DeLucia and Cairns (10) + Cairnsa 3 Cairns' + Cairnsc 12 Monk and Kinross (30) Monk and Kinross (30) + 1 Monk et al. (31) + Monk et al. (31) Hewitt et al. (23) + + This work. Mutant from ultravioletmutagenized C thy-321 1 E. coli C thy thyR HN9 This work. P1 transductant of HN6. Donor: JG138. E. coli C thy thyR HN10 This work. P1 transductant of HN6. + Donor: JG138. E. coli B thy thyR ton sul B/r thy Freifelder (13) + a polA+ revertant from P3478 selected on plates containing methyl methane sulfonate. b Temperature-insensitive derivative of strain P112. For derivation of P112, see reference 4. C polA+ revertant from P112tr selected on plates containing methyl methane sulfonate. + 1
droxymethyl)aminomethane (Tris)-salts medium base and Tris minimal medium were as described previously (32). Thymine was added at 2 pg/ml. In some experiments Casamino Acids (Difco, vitamin free) was added to 0.1%. Nutrient agar was 2.3% nutrient agar (Difco) supplemented with 0.25% NaCl. Nutrient soft agar was 0.8% nutrient broth (Difco) containing 0.25% NaCl and solidified with 0.7% agar (Difco). NET buffer consisted of 0.01 M Trishydrochloride, 0.1 M NaCl and 0.1 M ethylenediaminetetraacetate (EDTA), pH 8.1. Unless otherwise specified, cells were grown in flasks at 37 C with reciprocal shaking (ca. 60 cycles/ min). The culture volume was less than 20% of the total capacity of each flask. Change of media was accomplished by harvesting the cells on a Millipore HA filter (0.45-jum pore size). The cells were rinsed with Tris-salts medium base and suspended in the desired medium. Assay of viable cell concentration was accomplished by appropriate dilution of samples in Trissalts medium base and plating on nutrient agar plates using the soft-agar overlay method (3 ml of soft agar per plate). Radioistopic labeling of cellular DNA. An overnight culture was diluted 20- to 40-fold into a small volume (usually 1 or 2 ml) of radioisotopic labeling medium, and cells were allowed to grow to a cell density of 3 x 108 to 5 x 108/ml. The culture was diluted 10-fold with nonradioactive medium and allowed to grow for about one generation. The resulting mid-log-phase cells were then transferred to the desired medium for each experiment. The labeling medium was Tris-minimal supplemented with either [14Clthymine (1 juCi/ml, 2.1 lAg/ml) or ['HIthymine (20 pCi/ml, 2.1 ug/ml). Sedimentation of DNA through alkaline sucrose gradIents. A culture to be analyzed was chilled in an ice-water bath, and cells were collected by centrifugation at 7,000 x g at 4 C for 3 min in a Sorval RC2-B
centrifuge, washed once with chilled NET buffer, and finally suspended in chilled 15% (wt/vol) sucrose in NET buffer at a density of 10' to 1.5 x 10'/ml. (For comparison of two separate cultures labeled with '4C and 'H, respectively, they were mixed after being chilled prior to Sorval centrifugation.) A 0.1 volume of lysozyme solution (Boehringer, 20 mg/ml in NET buffer) was added, and the mixture was kept in an ice-water bath for 15 min. A 0.02-ml volume of the resulting spheroplast suspension (containing 2 x 107 to 3 x 107 spheroplasts) was then gently mixed into 0.1 ml of a lysing solution (15% [wt/volI sucrose-i M NaOH-0.01 M EDTA) layered on top of a 4.85-ml linear alkaline sucrose gradient (20 to 33% [wt/voll sucrose-0.5 M NaOH-0.5 M NaCl-0.005 M EDTA). After standing at room temperature for 15 to 30 min, gradients were centrifuged at 36,000 rpm at 20 C for 120 min in an SW39L rotor. About 32 fractions were collected dropwise from the bottom of the tube, and the acid-insoluble radioactivity in each fraction was assayed as described by Hanawalt and Cooper (21). This type of sucrose gradient was calibrated by using phage T6 DNA labeled with 140C]thymine, and confirmed to be practically isokinetic. Number average molecular weight (Mn) of DNA in the gradient was calculated with the equation: Mn=
if,
Z(fJMj
where f, is the percentage of radioactivity in fraction i, and M, is the molecular weight of a homogeneous DNA that would have its maximal concentration in fraction i. For the sake of reproducibility, fractions 9 through 28 were used for the calculation of average molecular weight. M, was obtained by the equation (1):
D, D2
(M) 0.38 \M2/
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539
which relates the molecular weights of two DNA preparations (M1 and M2) to the distance sedimented (D, and D,). "4C-labeled T6 DNA was used as a reference. Chemicals. [3H]thymine and [14C]thymine were purchased from New England Nuclear. Chloramphenicol, rifampin, and 5-methyltryptophan were from Calbiochem. Puromycin was obtained from Nutritional Biochemicals Corp.
RESULTS
Generality of thymineless-induced strand breaks and effect of polA mutation. In view of the conflicting reports as to the occurrence of thymineless-induced strand breaks in the chromosomal DNA of E. coli, we first examined sedimentation patterns of DNA from thyminestarved cells of various E. coli strains. Particular emphasis was placed on polA- strains, since one such mutant, P3478 polAl, has been reported to be unusually sensitive to thymineless death (38, 40; H. E. Bendigkeit and P. C. Hanawalt, Bacteriol. Proc., p. 36, 1968). Recently, Berg and O'Neill (5) reported similar results with polA6 as well as polAl mutants. Figure 1 shows typical results for strains W3110 thy- and P3478. In strain W3110 thy-, there was a very slow but progressive change in the DNA sedimentation pattern during a 180min period of thymine starvation. Thus, the peak became broader with time, and the position of the peak fraction shifted considerably toward the top of the gradient. Despite the apparent change in sedimentation profile, the average molecular weight of single-strand DNA decreased only by a factor of 1.6. In contrast, in strain P3478 the change in the shape of the sedimentation pattem occurred earlier (already apparent at 60 min), and the number of strand breaks for all time points examined was greater than that in strain W3110 thy-. After 180 min of thymine starvation, the reduction of average molecular weight was by a factor of 2.2. Strain PR3478, a poUA+ revertant from strain P3478, was indistinguishable from strain W3110 thyin this type of experiment (data not shown). This effect of the polAl allele was also confirmed by using a transductant pair, JG138 and JG139, although the accumulation of strand breaks was much less pronounced in strain JG139 polA+ than in strain W3110 thy-. In spite of the fact that these were apparently the same strains used by Sedgwick and Bridges (38), we have not been able to confirm their inability to find strand breaks induced by thymineless incubation. To ascertain whether the enhanced accumulation of strand breaks in polAl strains was actually the consequence of DNA polymerase I
'. 7.
cX 10 _
5
,, 1 120
00
I
.0~~~~~~~
10 12 3mi 0~~~~~~~. 50~~~~~~~~~~~~~~~~~1
Q 00
10
20 Froction No.
30
FIG. 1. Alkaline sucrose gradient sedimentation profiles of DNA from thymine-starved and nonstarved cells of strains W3110 thy- and P3478. Cells were labeled with [3H]thymine (strain W3110) or ["4C]thymine (strain P3478) and starved for thymine for the times indicated. The control (0 min) consisted of cells suspended in thymineless medium but kept in an ice-water bath until analyzed. The two cultures were then mixed and analyzed. Symbols: 0, strain W3110 thy-; *, strain P3478. Number average molecular weight for each profile is: strain W3110 thy-, 1.4 x 10' (0 min), 1.2 x 10' (60 min), 1.1 x 108 (120 min), and 8.9 x 107 (180 min); strain P3478, 1.3 x 10' (0 min), 6.7 x 107 (60 min), 6.3 x 107 (120 min), and 5.9 x 107 (180 min). The peak fraction for the both controls (fraction no. 14) corresponds to 3.4 x 10' molecular weight. Approximate survival level for each starvation period was: strain W3110 thy- 110%o (60 min), 50% (120 min), and 10%o (180 min); strain P3478, 70%o (60 min), 1% (120 min), and 0.2%o (180 min).
deficiency, the effects of two other polA mutations, polA12 and polA3, were examined. Figure 2 illustrates the results obtained with strains MM383 polA12 and MM384 polA+. Since a polA12 mutant produces a thermolabile DNA polymerase I (30), thymine starvation was carried out at both permissive (30 C) and nonpermissive (43 C) temperatures. Strain MM384 exhibited little, if any, change in sedimentation profile at 30 C for up to 150 min, but showed
NAKAYAMA AND HANAWALT
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J. BACTERIOL.
2 0
0
Z, t a 10 c)
w
Froction No.
FIG. 2. Alkaline sucrose gradient sedimentation profiles of DNA from thymine-starved and nonstarved cells of strains MM383 and MM384. Cultures were labeled with [9H]thymine (strain MM384) or [14C]thymine (strain MM383) and then starved for thymine at 30 or 42 C for the times indicated. The culture media included Casamino Acids. The two cultures were mixed and analyzed. Symbols: 0, strain MM383; 0, strain MM384.
substantial broadening and shift of the peak at 42 C. In strain MM383, on the other hand, a change in sedimentation pattern was evident even at 30 C, and at 42 C, it was much greater than that in strain MM384. The polA3 mutation also exhibited a similar enhancing effect on the accumulation of thymineless-induced strand breaks in experiments with strains P112tr and PR112tr (data not shown). Strain PR112tr showed only slight change in sedimentation profile during 240 min of thymine starvation (in the presence of Casamino Acids). The above results were obtained by comparing strains in pairs, each of which was almost isogenic except for the polA locus. Furthermore, three independent polA mutations exerted a similar enhancing effect on the accumulation of thymineless-induced strand breaks. Since polA12 is considered to be a missence mutation, a contribution to the present phenomenon by a possible polar effect of the polAl amber mutation on adjacent genes may be eliminated. Therefore, we conclude that the observed effect on thymineless-induced strand breaks is actually due to the deficiency in DNA polymerase I. Baker and Hewitt (2) reported almost no strand breaks in thymine-starved E. coli C thy-321. We reexamined this same strain to find that it behaved in a unique manner. The DNA sedimentation profile in this strain broadened at an early stage of thymine starvation (30 and 60 min), then returned completely to the original shape (120 min) and remained that way until 240 min (Fig. 3). By 300 min, however, a
small number of strand breaks seemed to have reappeared. This early change in the sedimentation pattern was reproducible, although it had not been seen by Baker and Hewitt (2). The subsequent restoration of the normal profile seemed to be in agreement with their observations. Figure 3 shows that the polAl mutation enhances the accumulation of strand breaks in this strain as well. Strain B/r thy-, a representative of B series strains,' was also tested and found to exhibit about as many strand breaks as strain P3478 under the same conditions (data not shown). Effects of metabolic inhibitors on thymineless-induced strand breaks. The experiments described so far have established that strand breaks accumulate in the chromosomal DNA of thymine-starved cells in the majority of strains tested. The next aspect concerns the mechanism for their formation and the possibility of a causal relationship between strand breaks and thymineless death. The effects of various metabolic manipulations on cells undergoing thymineless death were studied. The results are summarized in Fig. 4, and the following statements may be made within the limits of these experiments. In strain W3110 thy-, the formation of thymineless-induced strand breaks seems to be almost completely blocked by energy deprivation (glucose starvation or cyanide), starvation for inorganic phosphate, inhibition of protein synthesis (chloramphenicol, 5 ,ug/ml or higher; puromycin; and 5-methyltryptophan) or ribonucleic acid synthesis inhibition (rifampin).
I
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DNA STRAND BREAKS AND THYMINELESS DEATH
In strain P3478 polA -, on the other hand, only deprivation of energy source (glucose starvation) and ribonucleic acid synthesis inhibition (rifampin) were as effective in preventing thymineless-induced strand breaks as in strain W3110 thy-. (Since thymine starvation in the presence of 0.01 M NaCN caused substantial cell lysis in strain P3478 for unknown reasons, the effect of cyanide could not be evaluated.) Starvation for inorganic phosphate and chloramphenicol (even at 25 Ag/ml) exerted only a partial prevention of strand break accumulation, whereas puromycin showed only little, if any, effect in strain P3478. Rejoining of thyminless-induced strand breaks. Thymineless-induced strand breaks can be rejoined when thymine is added back to a starved culture. Figure 5 shows the results of an experiment in which strains P3478 and PR3478 were starved for thymine for varying lengths of time and then allowed to grow in the presence of thymine for 15 min. The DNA was analyzed in alkaline sucrose gradients before and after the 15-min incubation period with thymine. Although each set of thymine starvation periods was primarily chosen so that the two strains give similar survivals, they resulted in comparable sedimentation profiles as well
(i.e., similar numbers of strand breaks) for DNA from both strains. The examination of postincubation sedimentation patterns has revealed obvious rejoining of the strand breaks, as evidenced by restoration of a normal or nearly normal profile, but no evident difference between wild-type and polA strains. This suggests that the polA mutation does not grossly affect the rate of rejoining of the strand breaks after restoration of thymine. It must also be pointed out that, although return to the normal sedimentation profile effected by the 15-min incubation period with thymine was nearly complete after 180 min (for strain PR3478) or 90 min (for strain P3478) of thymine starvation, the survival level under these conditions was only about 10% (see below and Fig. 7 and 8). Survival studies. To clarify the relationship between thymineless-induced strand breaks and thymineless death, the effects upon viability of the polA mutation and the various metabolic treatments were examined. As clearly seen in Fig. 6 each of three polA alleles rendered the cells hypersensitive to thymineless death. It is noted that strain MM383 died faster than strain MM384 even at 30 C, a permissive temperature. This finding, consistent with the analysis of
[_S
0
541
'sI
0
C.) 0
0
.:
KZ
Fraction No.
3. Alkaline sucrose sedimentation profiles of DNA from thymine-starved and nonstarved cells of strains C thy-321 and HN9. Cells were labeled with [3H]thymine (strain C thy-321) or [14C]thymine (strain HN9), thymine-starved for various lengths of time, and then analyzed. The experiment with strain HN9 has been done with 'IC-labeled, thymine-starved or nonstarved cells of strain HN10 as internal controls. Strain-HN10 behaved in exactly the same way as strain C thy-321. Left, Strain C thy-321; right, strain HN9. Symbols: *, nonstarved; 0, starved for 30 min; A, 60 min; A, 120 min; *, 180 min; 0, 240 min; and *, 300 min. FIG.
J. BACTERIOL.
NAKAYAMA AND HANAWALT
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-.1
a 4-
0 .O
0
V0
10
20
30
10
20
30
10
20
30
Fraction No. FIG. 4. Effects of various treatments of DNA sedimentation profiles of thymine-starved cells. Cells of strains W3110 thy- and P3478 were labeled with [3H]thymine and then starved for thymine for 180 min (strain W3110 thy-) or 120 min (strain P3478) with or without various treatments. Each treatment was introduced simultaneously with thymine deprivation and continued throughout the starvation period. Each experiment was monitored by including appropriate internal control (not shown in the figure) labeled with [14Clthymine (nonstarved cells or cells thymine-starved without additional treatment). Symbols: 0, strain P3478; 0, strain W3110 thy-. (a) Nonstarved; (b) thymine starved (- 7T); (c) T, starved for glucose; (d) T, NaCN (0.01 M), (e) T, starved for phosphate; (f) T, chloramphenicol (25 Ag/ml); (g) T, 5-methyltryptophan (200 ug/ml); (h) -T, puromycin (2,000 gg/ml); (i) -T, rifampicin (30 Ag/ml). In experiments with rifampin, cells were pretreated with EDTA according to Rose et at. (37), except that the drug was added immediately after dilution following EDTA treatment. A control experiment showed that this conditioning of the cells did not influence the accumulation of thymineless-induced strand breaks. -
-
-
strand breaks (Fig. 2), suggests that some DNA polymerase I deficiency is manifest in this strain even at the permissive temperature. It is also worthy of note that in strain PR112tr thymine starvation for 240 min, which caused only few strand breaks, resulted in significant killing, survival being about 10%. The effects on thymineless death of various metabolic treatments in strains W3110 thyand P3478 are presented in Fig. 7 and 8. In strain W3110 thy-, cyanide, absence of glucose or inorganic phosphate, chloramphenicol (25
-
-
lsg/ml or higher), puromycin, and rifampin have nearly eliminated thymineless death. Those effects appear to agree with the almost complete inhibition by the same treatments of thymineless-induced strand breaks. In contrast, however, chloramphenicol at a lower concentration (5 Ag/ml) and 5-methyltryptophan are only partially effective in preventing thymineless death in spite of their virtually perfect blocking of the induction of strand breaks. In strain P3478 only glucose starvation resulted in almost complete prevention of thymineless death. Re-
VOL. 121, 1975
DNA STRAND BREAKS AND THYMINELESS DEATH
0o
543
Cwc) l Cd)
10
0 0
10 iW/\
10
20
3 Fraction No.
10
20
30
FIG. 5. Rejoining of thymineless-induced strand breaks. Cells of strains PR3478 and P3478 were labeled with
[3 H)thymine and [14C]thymine, respectively, and then starved for thymine for various times. Thymine was added back to each culture at 2 1gg/ml, and further incubation was made for 15 min. Sampling was carried out
before and after the incubation with thymine. Samples of strains PR3478 and P3478 were combined, as indicated below, soI0that survivals for the two cultures mixed were approximately the same, and analyzed. Symbols: O, strain P3478; *, strain PR3478. (a) Thymine starved for 120 min (PR3478) or 60 min (P3478); (b) thymine starved as in (a) and incubated with thymine for 15 min; (c) thymine starved for 180 min (PR3478) or 90 min (P3478); (d) thymine starved as in (c) and incubated with thymine for 15 min; (e) thymine starved for 250 min (PR3478) or 120 min (P3478); (f) thymine starved as in (e) and incubated with thymine for 15 min. Approximate survivals were 50, 10, and 1% for (a), (c), and (e), respectively.
10-2
0
60 120
180 240 0 6 20 Time of Thymine Storvrtion (min)
180
240
FIG. 6. Thymineless death at various polA8tand polA + strains. Symbols:P, (a) strain W3 10 l, strain thyw; i tr; , strain Pw12 tr. (b) S, strain MM384 ats 30 C; , strain MM383 at 30oC; , strain , strain PR112 MM384 at42bC; , strain MM383 at The culture media used in the experiments involving P112tr 42sC. PR1 12tr, MM383, and MM384 contained Casamino Acids.
P34b78;
544
NAKAYAMA AND HANAWALT
