In vitro replicative arrest in dnaAts mutants

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 190, No. 2, October, 671-676,1978

In Vitro Replicative MUSETTA

Arrest in dnaA,, Mutants

A. HANSON’

AND

ROBB E. MOSES

Baylor College of Medicine, Department of Cell Biology, Houston, Texas 77030 Received February

21, 1978; revised May 10, 1978

Toluene-treated Escherichia coli, conditionally defective in initiation of DNA replication, have been studied. Toluenized mutants of the dna& class rapidly stop DNA synthesis at the restrictive temperature. This is in contrast to the slow arrest of replicative synthesis noted in vivo in these strains. The in vitro cessation of replicative DNA synthesis can be prevented by the presence of the detergent Triton X-100. Our results suggest a role in elongation of DNA by the dnaA gene product during replicative synthesis in vitro. INTRODUCTION The control of DNA replication has been studied by the use of temperature-sensitive mutants. There are two main classes of these mutants, “slow-stop” initiation and “fast-stop” elongation mutants (1). The initiation mutants are thought to finish rounds of DNA replication already begun but not to initiate new rounds when shifted to the restrictive temperature. This is in contrast to the elongation mutants in which synthesis ceases rapidly upon shift to restrictive temperature. The behavior of the initiation mutants, dnaA and dnaC, at the restrictive temperature is similar to that seen in wild-type cells inhibited in protein synthesis. The quantity of DNA made in the initiation mutants at restrictive temperature agrees with that expected for completed rounds of replication and is similar to the amount made when wild-type cells are held in chloramphenicol (2-4). dnaA mutants incubated at restrictive temperature, and wild-type cells arrested in DNA synthesis by amino acid starvation, have membrane proteins with an altered electrophoretic mobility (5, 6); furthermore, changes in cell surface properties appear to be associated with the arrest of initiation. dnaA cells are abnormally sensitive to lysis by deoxycholate when incubated at restrictive temperature, and bind an aniline dye with unusual efficiency. These changes do not occur in dnaB or wild-type cells restricted in DNA synthesis (3). These observations suggest a membrane alteration in dna.A cells. Replicative DNA synthesis occurs in vitro in toluene-treated E. coli cells (7) in a manner similar to duplicating intact cells. The in vitro system allows definition of an ATP requirement for replication. However, there is no evidence for initiation of new rounds of replication in this system (8). Mutants of the “fast’ Present address: National Bethesda, Md. 20014.

Institutes

0003-9861/78/1902-0671$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

stop” variety (dnaB,) show an arrest of DNA synthesis in 3-5 min at the restrictive temperature in vitro, in agreement with their in vivo behavior. Mutants of the initiation type have been thought to show no defect at the restrictive temperature in the toluenetreated cell system, because reinitiation does not occur

in vitro. This paper presents data which show that in dna& mutants replication unexpectedly ceases rapidly in toluene-treated cells at the restrictive temperature (42”), in contrast to another initiation mutant, dnaCtS. This behavior suggests a defect in DNA chain elongation at the restrictive temperature and a pleitropic nature of the dnaA gene. Using the toluene-treated cell system, we have observed that plateau stage wild-type E. coli cells show diminished replication in vitro and that the loss of synthesis can be reversed by the presence of Triton X-100 (9). Our observations prompted us to investigate the effect of the detergent on DNA synthesis in toluene-treated initiation temperature-sensitive cells assayed at their restrictive temperature. We find that the arrest of synthesis at 42” in vitro in dnaA, mutants is prevented if Triton X-100 is included in the reaction mixture, but that Triton X-100 has no effect on levels of synthesis occurring in dnaC initiation mutants or dnaB elongation mutants.

MATERIALS

AND

METHODS

Preparation of toluene-treated cells and strains used. E. coli cells were prepared as previously described (7). Cultures were grown in L-broth supplemented with thymine (1 &ml). At a cell concentration of 5.0-7.0 x lo8 cells per ml, the cultures were harvested by centrifugation at 4°C at 8000 rpm for 10 min. Cells were resuspended in 0.05 M KPOI, pH 7.4, to a volume 5% of the original culture volume. Toluene (1%) was added, the suspension agitated at 25°C for

of Health, 671

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10 min, and then centrifuged as above. The pellet was again resuspended in 5% the original volume, and these cells used in the assay for DNA synthesis. The strains used are summarized in Table I (1). Assay of DNA synthesis. The assay for synthesis was as previously described (9). The reaction mixture (0.3 ml) contained 70 mM KPOd buffer, pH 7.4; 13 mM MgClz; 1.3 mru ATP, 33 PM dCTP, dGTP, dATP, [3H] dTTP; and 2 x 10’ toluene-treated cells. Incubations were done at 32°C and 42°C. When Triton X-100 was used it was at a concentration of 1% and 10 mru 2mercaptoethanol was added (9). Reactions were terminated by adding 3 ml cold 10% trichloroacetic acid, 0.1 M sodium pyrophosphate. The precipitates were collected on Whatman GF/C glass filters and washed three times with cold 10% TCA’ and once with cold 0.01 M HCl. The filters were dried and radioactivity determined by liquid scintillation. Isopyknic analysis DNA synthesis in vitro was analyzed using density label transfer to determine the mode of synthesis. E. coli were grown with a prelabel of [‘?]thymidine (1 pCi/ml), and harvested at various stages of growth. The cells were then toluene-treated in the usual manner and added to an assay mixture containing [3H]dATP and BrdUTP as a density label in place of dTTP (10). The reaction was stopped with 2 ml NET buffer (0.1 M NaCl, 10 mru EDTA, 10 mM Tris, pH 8.0) and the cells collected by spinning at 15,000 g for 15 min. The pellet was resuspended in NET/10 buffer with pronase (1 mg/ml) and 1% Sarkosyl and allowed to lyse at 60” C for 2 h. Following lysis, 65% CsCl was added to each tube to a volume of 8.0 ml and centrifugation carried out for 60 h at 37,000 rpm at 25°C in a Beckman 50 Ti rotor. The gradient was collected on Whatman 3MM paper strips, washed with cold TCA, dried, and counted in a liquid scintillation counter. RESULTS

dnaA, Mutants in Vitro Conditionally defective initiation mutants of E. coli, dnaA and dnaC, are characterized by a slow stop of DNA synthesis following a shift to restrictive temperature in vivo. Cessation of replication at the restrictive temperature requires approximately one generation. The dna& strain, CRT 4636, shows typical in vivo kinetics of incorporation at the restrictive temperature (Fig. 1). This has been interpreted as completion of rounds of replication, but failure to initiate new ones (1). In contrast, when dnaA, cells, grown at permissive temperature, are toluene-treated and assayed in vitro for DNA synthesis at restrictive tem’ Abbreviation

used: TCA, trichloroacetic-acid.

MOSES TABLE

I

BACTERIALSTRAINS Strain

Genotype

CRT 4636 CRT 46 PC 22 E 177 E 508 JW 200 w3110 P3478

POW, drm.4, dna.L

POW, dnaG dnaAts dna& POLK, dnaB, wild-type

POLK

0 0

20 40

Source Y. Hirota Y. Hirota Y. Hirota J. Wechsler J. Wechsler J. Wechsler J. Cairns J. Cairns

60 SO 100 120 M/N

FIG. 1. In vivo DNA synthesis by CRT 4636 (PO&I-, dnaA,). [3H]Thymidine (1 @i/ml) was added to a culture of cells (at 0 min) grown at 32°C to a density of 1 X 10s cells/ml. The culture was split in half and one-half was incubated at 32’C, the other halfat 42°C. At regular time intervals, samples were removed from each, precipitated with cold 10% trichloroacetic acid, 0.1 M sodium pyrophosphate, filtered, dried and counted by liquid scintillation.

perature, DNA synthesis ceases in about 5 min (Fig. 2). This rate of arrest is surprising for a mutant supposedly deficient only in initiation and suggests a defect in elongation in the in vitro system at restrictive temperature. The same observation has been made with dna& strains E177, E508, and CRT46, all showing arrest of DNA synthesis in vitro in about 5 min. These results suggest that toluene-treatment of these mutants sensitizes a component re-

cZnoA, MUTANT

0510

20

30 0 5 rfhw

10

20

in Vitro

30

(MIN)

FIG. 2. In vitro DNA synthesis by CRT 4636 (PO&, &z&J. Cells were grown at 32”C, toluene-treated and assayed as described. The assays were carried out in ATP (1.3 mM) at 32’C and 42°C and in the presence and absence of Triton X-100. Incorporation of [3H] dTMP into acid-precipitable material was followed.

quired for replicative DNA synthesis in vitro. The in vitro results of the dnaA, strains are in contrast to that of another initiation mutant which has similar in viuo kinetics. A dnaG mutant (PC22), grown at permissive temperatures, when toluene-treated and assayed in vitro at 32°C and 42”C, shows a pattern of a slowly decreasing rate of synthesis at the restrictive temperature (Fig. 3A), as opposed to the more rapid arrest of the dnaA mutant (Fig. 3B). The slight decrease in the rate of synthesis at 42°C seen in the dna& mutant is no greater than the decrease seen at 42°C using wild-type cells. Furthermore, Triton X100 has no effect on the level of synthesis (data not shown), in contrast to the data obtained with a dna.A, mutant (Fig. 2). For dnaAti mutants Triton X-100 prevents the tion of synthesis at 42’C and allows ATPdependent DNA synthesis to proceed at the level found in wild-type cells. As noted in Fig. 2, Triton X-100 has no effect on the level of DNA synthesis at permissive temperature. The results with dna& cells are also in contrast to those found in another temperature-sensitive strain, JW 200, an elongation mutant ( dnaB,, poZA1). Incorporation in toluene-treated log phase cells of JW 200 is linear with time at 32°C but, when the cells are shifted to the restrictive temperature (42”C), synthesis ceases rapidly (3-5 min) and is not protected by Triton X-100 (9).

REPLICATIVE

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Isopyknic Analysis Density transfer analysis was used to determine the mode of DNA synthesis in the dnaA, cells in uitro. Panels A and B (Fig. 4) demonstrate that Triton X-100 does not significantly alter the characteristics of replicative synthesis at the permissive conditions; the synthesis seen is primarily semiconservative as indicated by the synthesized DNA of hybrid density. At 42’C, without Triton X-100 (panel C), there is little semiconservative synthesis, indicating that replication is very limited at the restrictive temperature in vitro. This supports the kinetics shown in Fig. 2. In the presence of Triton X-100 (panel D), semiconservative synthesis is maintained at levels similar to those seen under permissive conditions. Inasmuch as Triton X-100 was present throughout the incubation at restrictive temperature, it appears that the detergent prevents arrest of semiconservative DNA synthesis in vitro. Panels E and F indicate that in the absence of ATP, no replicative synthesis occurs, independent of the presence of Triton X-100. This is also true at 32°C (data not shown). The absence of an increase in the light density peak (panels B, D, and F) in the presence of Triton X-100 with or without ATP at 32°C or 42’C argues that the detergent is not stimulating a

FIG. 3. In vitro DNA synthesis by PC22 (POW, cZn&J and CRT 4636 (POW, cZnu.4,). Cells were grown, toluene-treated and assayed at 32°C and 42°C. as described in Fig. 2.

674

HANSON

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42’- ATP - fiiton ‘. ‘.,, g/cc ‘,‘~

fR4CUON

AND

f c

MOSES

5 _, 42” ‘, ‘,.

ATP+ ?kitm ^

g/cc

IO

NUMBER

FIG. 4. CsCl density gradient analysis of in vitro DNA synthesis in CRT 4636 (POLK, &a&). Cells were prelabeled with [Wlthymidine (0.1 pCi/ml), grown at 32”C, and toluene-treated as previously described. The assays (as in Materials and Methods) were carried out with C3H]dATP (150 cpm/pmol) and BrdUTP as a density label in the presence and absence of Triton at 32°C and 42°C for 30 min. The cells were lysed, the gradient prepared, collected, and analyzed as described in Materials and Methods.

repair or nonspecific DNA synthesis. Because semiconservative DNA synthesis is maintained at 42°C in the presence of the detergent, it seems that Triton X-100 protects some facet of replication which is thermolabile after toluene-treatment in this mutant. DNA polymerase I does not alter this observation because the same results are obtained in dnaA, strain containing polymerase I (CRT 46). The same results also have been obtained with independently isolated dnaA, mutations (E177, E508), for both mode and extent of synthesis. Analysis

of Revertants

In order to ensure that the arrest of synthesis in vitro is indeed due to a dnaA gene mutation, spontaneous revertants of CRT

4636 were selected by growth at the restrictive temperature and analyzed for synthesis at the permissive and restrictive temperatures. DNA polymerase assays were performed on extracts of the revertants to confirm that they remained poZA-. No cessation of synthesis at 42°C was observed in vivo in strains selected. There is no shut-off of synthesis at 42°C in such revertants in vitro (Fig. 5). The slight decrease in the rate of synthesis is equal to that seen when wild-type cells are assayed at 42°C in an identical manner. Isopyknic analysis of synthesis in the revertant at permissive and restrictive temperatures shows the product in both cases primarily is made semiconservatively (Fig. 6). Furthermore, Triton X100 has no effect on the levels of synthesis (data not shown). Thus the thermosensitiv-

&oA,

MUTANT

CRT4636 rev1 fpo/A-j

in Vitro REPLICATIVE

675

ARREST

, 3f

10

P

-d

5

% I.2 ‘.a

42’ x

z 0

x

42’+ATP

z u

‘\ \

‘= _ IO -c

5

FRACTION I

I

0 5 10

I

1

20

30

T/ML= /MN) FIG. 5. In vitro DNA synthesis by CRT 4636 rev 1 (polA-). Cells were grown, toluene-treated, and assayed as previously described. The assays were carried out at 32°C and 42’C and incorporation of [3H]dTMP into acid-precipitable material followed as described in Fig. 2.

ity of semiconservative DNA synthesis following toluene-treatment appears to be due to the dna.A gene mutation conferring in uiuo thermosensitivity. DISCUSSION

Our results present two different phenomena. First, dnc& mutants are thought to be initiation-defective and display “slowstop” kinetics in ho. However in the toluene-treated cell system at restrictive conditions these mutants show “fast-stop” kinetics similar to those seen in the elongation mutants. This suggests a possible elongation function in replication for the dnaA gene product, as well as the reported function in initiation. Novobiocin, a drug inhibiting elongation of DNA strands during replication, appears to act on the product of a gene tightly linked to dnuA (11). This underscores the need for evaluating a possible role of dnah in elongation. It appears

NUMBER

FIG. 6. CsCl density gradient analysis of in vitro DNA synthesis in CRT 4636 rev 1 (polA-). Cells were grown, toluene-treated, assayed, and the gradients prepared as described in Fig. 4.

the in vitro “fast-stop” behavior of mutants is the result of toluene treatment, perhaps due to the sensitization of a structure involving the dnaA gene product. However, the effect is specific for the dnaA mutation, as nontemperaturesensitive revertants do not show in vitro arrest (Fig. 5). An important contrast is that dnaCt, mutants, another class of initiation mutants, do not show rapid in vitro arrest. Arguments have been presented that the dnaA gene product is a membrane component required for initiation (5, 6). Hehnstetter (11) has proposed a model of replication which calls for two membrane attachment sites; one site attaches the DNA helix to the membrane while the other site attaches to the replication machinery. If the dnaA gene product were a pleiotropic protein involved in the structure of the second attachment site, the in vitro faststop behavior might be explained on the basis of disrupted membrane attachment. In non-toluene-treated cells, the loss of elongation function might not be observed because there is no perturbation of the that

dna&

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HANSON

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membrane. However, the initiation function of the mutated dnaA protein could be impaired at high temperature resulting in “slow-stop” behavior in vivo. The second phenomenon which we observe is that Triton X-100 prevents replicative arrest in vitro in dnuA, mutants. It may be that the detergent prevents or reverses an in vitro sensitization of a hydrophobic protein, in effect stabilizing the replication attachment site against denaturation. Alternatively, the detergent might be able to substitute for a membrane component required for replication. Using the toluene-treated cell system, we have previously reported that plateau stage wild-type E. coli celIs show diminished replication in vitro (compared to log phase cells) and that the loss of synthesis can be reversed by the presence of Triton X-100 (9). The restored synthesis is semiconservative in nature, and temperature-sensitive in dnaB, mutants, but whether it is initiated at an unique site is uncertain. These experiments used an in vivo arrest of DNA replication, which could be reversed in toluene-treated cells by Triton X-100. Hanna et al. (13) have reported a reversal in toluene-treated cells by Triton X-100 of the arrest of synthesis following an incubation at the restrictive temperature in vivo of an initiation defective (dnaCJ mutant. Harper, et al. (14) have further characterized the DNA synthesis stimulated by Triton X100. They suggest that Triton-stimulated synthesis has both repair and replicative aspects. In addition to being semiconservative, there is an apparent requirement for the recB gene product and a nonspecific ATP requirement for synthesis. In considering this in relation to our data, it is important to note that the experiments reported here deal with a completely different situation-arrest of DNA synthesis by an in vitro shift to restrictive conditions after toluene-treatment. In the other works cited

MOSES

above, Triton X-100 reverses in vitro a physiological arrest of DNA replication in vivo due to stationary phase or restrictive temperature. There is nothing to indicate that the protective action of the detergent described in this paper and the reversal of arrest by the detergent described in the other publications operate by the same mechanism. The failure of Triton X-100 to prevent replicative arrest in a mutant of the elongation type shows that the effect is not a general one. ACKNOWLEDGMENTS This work wee supported by National Institutes of Health Grant GM-19122 and Robert A. Welch Foundation Grant No. Q-543. M.A.H. was supported by National Institutes of Health Grant GM-05421. REFERENCES

1. GROSS, J. D. (1972) Curr. Topic. Microbiol. munol. 57,39-74.

Im-

2. BEYERSMANN, D., SCHLECHT, M., AND SCHUSTER, H. (1971) Mol. Gen. Genet. 111,145-158. 3. HIROTA, Y., MORDOH, J., AND JACOB, F. (1970) 2

Mol. Biol. 53,369-367. 4. KUEMPEL, P. L. (1969) J. Bacterial. 100, 13021310. 5. SHAPIRO, B., SICCARDI, A., HIROTA, Y., AND JACOB, F. (1970) J. Mol. Biol. 52, 75-89. 6. SICCARDI, A., SHAPIRO, B., HIROTA, Y., AND JACOB, F. (1971) J. Mol. Biol. 56,475-490. 7. MOSES, R. E., AND RICHARDSON, C. C. (1970)

Proc. Nat. Acad. Sci. USA 67.674-681. 8. BURGER, R. M. (1971) Proc. Nat. Acad. Sci. USA 68,2124-2126. 9. MOSES, R. E. (1972) J. Biol. Chem. 247, 60316038. 10. MOSES, R. E., AND MOODY, E. (1975) J. Biol.

Chem. 250,8055-8061. 11. RYAN, M. J. (1976) Biochemistry 15,3769-3777. 12. HELMSTEITER, C. (1974) J. Mol. Biol. 64,21-36. 13. HANNA, M., SOUCEK, L., AND CARL, P. (1975) in DNA Synthesis end Its Regulation (Gouhen, M., end Henawelt, P., eds.), W. A. Benjamin, Inc., Menlo Park. 14. HARPER, D. J., CHEN, P. L., AND CARL, P. L.

(1977) Biochim Biophys. Acta 474,363-377.