JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 755-762 0021-9193/78/0133-075502.00/0 Copyright X 1978 American Society for Microbiology

Vol. 133, No. 2 Printed in U.S.A.

dnaA Acts Before dnaC in the Initiation of DNA Replication FANG-CHIEN KUNG AND DONALD A. GLASER* Department of Mokcular Biology, University of California, Berkeley, California 94720

Received for publication 21 November 1977

We constructed a double mutant of Escherichia coli K-12 carrying dnaA(Ts) and dnaC(Cs) lesions. In this mutant DNA synthesis proceeds normally at 320C and initiation is inhibited at both 41 and 200C. By shifting this culture grown at 320C to the two restrictive temperatures in different time sequences and assaying protein and DNA synthesis of cells growing at different temperatures, we found that dnaA and dnaC genes work independently with dnaA acting before dnaC. While preparing special strains for this work, we also showed that the order of genes in the neighborhood of dnaA is dnaA-tnaA-phoS-ilv.

The rate of DNA replication in Escherichia coli is regulated mainly by mechanisms that control the frequency of initiation of DNA replication. Once initiated, DNA replication starts at a fixed origin (4, 25) and proceeds bidirectionally (1, 17, 23, 25, 26) at a constant speed (11) to a terminus opposite the origin on the circular chromosome (28). The initiation process may require RNA (9, 12, 19, 23, 24) and protein synthesis (9, 12, 20). Also sufficient cell mass (7) or accumulation of enough "initiation potential" in a preinitiation period (11) seems to be essential for this process. Some negative control mechanisms for initiation have also been proposed by Rosenberg et al. (29) and Pritchard et al. (27). There is insufficient evidence to support or refute either positive or negative control for initiation of DNA replication. dnaA (2, 14, 35, 36), dnaC (2, 31, 34, 35, 38), dnaI (2), and dnaP (33) mutants are thought to be defective in initiation of DNA replication. There is new evidence showing that a dnaB mutant (dnaB252) may be an initiation instead of an elongation mutant (41). A new dnaM locus has been isolated recently, and it may also be associated with DNA initiation (31). Judging from the complexity of genetic control of DNA replication and the biochemical studies done mostly in the bacteriophage systems, it seems that the DNA initiation process is very complicated. Several cold-sensitive dnaA and dnaC mutants (34, 36) and a large number of heat-sensitive dna mutants (31) have been isolated in this laboratory. We constructed a double mutant carrying dnaC(Cs) of LW126 (34) and dnaA5 of PC5 (5). This mutant, FK8133, grew normally at 320C but could not reinitiate DNA replication at 20 or 410C. If dnaA and dnaC genes act independently and dnaA acts before dnaC, a

culture of FK8133 should be able to initiate a new round of DNA synthesis when shifted from 32 to 200C, allowed to complete rounds at that temperature, and then shifted to410C. [200C is a permissive temperature for dnaA(Ts) but a restrictive temperature for dnaC(Cs), whereas 410C is a restrictive temperature for dnaA(Ts) and a permissive temperature for dnaC(Cs).] With the same assumptions, a culture of FK8133 should not be able to initiate a new round of DNA synthesis when shifted from 32 to 410C, allowed time to complete rounds at 410C, and then shifted to 20°C. The experiments agree with these expectations and we conclude that dnaA and dnaC function independently, with dnaA acting before dnaC. In the process of strain construction we first mapped the cold- and heat-sensitive dnaA genes unequivocally. We find that the gene order of dnaA-tnaA-phoS-ilv agrees with that previously reported on the new E. coli chromosome-map (1). The adoption of this- gene order on the new map was based upon some statistically unconvincing data. In addition, the only other reported cold-sensitive dnaA gene suggested by Wehr et al. (36) did not agree with this gene order. We showed that the positions of temperature-sensitive dna lesion carried by PC5 and the coldsensitive dna lesion carried by CJ2 were very close, if not identical, and the relative position to the nearby genes was the same as previously reported: dnaA(Ts and Cs)-tnaA-phoS-ilv.


MATERIALS AND METHODS Bacterial strains. The bacterial strains and their genotypes are listed in Table 1. Phage Plvir was obtained from A. J. Clark. Medium and growth conditions. Solution K (18), pH 6.8, was used as minimal liquid medium. Required amino acids were added to solution K to a final concentration of 30,ug/ml, thymine was added to 10 ,ug/ml



KUNG AND GLASER TABLE 1. Bacterial strains Strain



BE280 AT2459 AT2255(DG17)

FHfr F-







PC5 FK1263




ilv-280 trp-37 tna-4 phoS3 lac-213 xyl-13 strA106 thi-1 serB22 rel-I Aleu lac his-i malA xyl argG metB thy-2 mtl gal ton str AdnaC(Cs) leu lac his-i malA xyl argG metB thy-2 mtl gal ton str AdnaA(Cs) leu lac his-I malA xyl argG metB thy-2 mtl gal ton str XdnaC659 leu lac his-i malA xyl argG metB thy-2 mtl gal ton str Athy leu str dnaA5 dnaC(Cs) leu lac his-i malA xyl argG metB thy-2 mtl gal ton str A- ilvD88 dnaA5 dnaC(Cs) leu lac his-I malA xyl argG metB thy-2 mtl gal ton str A-

when needed, and glucose was added to 0.2% (wt/vol). Minimal solid medium was prepared by using the 007 medium as described previously (36). Other nutrients were added at the same concentrations as in minimal liquid medium. For assaying DNA and protein synthesis in dna(Ts) mutants, cells were grown in a labeling medium composed of solution K supplemented with the following nutrients: Casamino Acids at 0.1% (wt/vol); thiamine, 1 ,ig/ml; glucose, 0.2%; thymine, 5 jig/ml; [methyl-83H]thymine, 0.5 MCi/ml (7 Ci/mmol from Schwarz/Mann); and [1-'4C]leucine, 5 tCi/ml (309 mCi/mmol from Schwarz/Mann). Luria (L) broth without CaCl2 was used as a complex medium. For preparation of phage P1, L medium was supplemented with 0.2% glucose, 10,ug of thymine per ml, and 2 mM CaCl2 (LCTG medium). L and LCTG plates were made of 2% agar in L or LCTG medium. Fresh overnight culture, grown in liquid medium at the permissive temperature without shaking, was diluted into fresh prewarmed medium to a final concentration of 1 x 107 cells per ml. Incubation was continued with shaking for 2 to 3 h to obtain a balanced and exponentially growing culture. Cell growth was monitored by measuring cell concentration with a Coulter Counter or with a Zeiss PMG II spectrophotometer. Preparation of phage P1 and transduction. A high-titer phage P1 stock was prepared by the confluent plate lysis technique as described by Wehr et al. (36). Phages were allowed to adsorb for 20 min at 37°C or 25 min at 35°C (for thermosensitive recipient bacteria) and unadsorbed phages were removed by centrifugation. Selection of temperature-sensitive mutant. Temperature sensitivity of the mutants was tested by their inability to form colonies at the restrictive temperature. Those that passed the test were then tested for DNA synthesis at the restrictive temperatures as described below. Radioactivity measurement. For the measurement of cellular DNA and protein synthesis at different temperatures, cells were inoculated into labeling medium and allowed to stand overnight at the permissive temperature. These uniformly labeled cells were

Source B. J. Bachmann B. J. Bachmann This lab

This lab This lab This lab This lab This work

This work

then reinoculated into labeling medium and incubated at the permissive temperature with shaking for at least 2 h to ensure balanced and exponential growth. To keep the culture in an exponentially growing phase, fresh medium equilibrated to appropriate temperatures was used for dilution during the course of longterm labeling. Samples of 50 Ad were taken at appropriate time points. The samples were treated as described previously (18). Enzyme assays. The activities of tryptophanase and alkaline phosphatase of each transductant in the mapping study were assayed in the following way. A 2-ml portion of L-broth culture grown overnight (more than 15 h) was used for both assays. The alkaline phosphatase assay was a modification of the method of Echols et al. (8). A 0.2-ml portion of a 0.5% solution of p-nitrophenyl phosphate in 1 M tris(hydroxymethyl)aminomethane, pH 8.0, was added to the culture and mixed. After 3 min of incubation, the culture of BE280 (phoS) turned to bright yellow and that of AT2255 (phoSg) did not change. These two cultures served as controls. Tryptophanase was assayed in the same culture tube used for the alkaline phosphatase assay. A 0.5ml portion of Kovacs' reagent (Difco Manual, p. 53, 9th ed., Difco Laboratories, Inc.) (5 g ofp-dimethylaminobenzaldehyde dissolved in 75 ml of amyl alcohol and 25 ml of concentrated hydrochloric acid) was added to the culture. A separated organic layer is formed above the aqueous phase. This organic layer of AT2255 (tna+) turned red immediately, whereas that of BE280 (tna) remained the original yellow color after mixing. Again these two cultures served as the control in this assay.

RESULTS Mapping of the dnaA gene. The dnaA gene has been mapped in the vicinity of the ilv operon, at about 82 min on the new genetic map ofE. coli. PC5 is a thermosensitive dnaA mutant (4). CJ2 is a new cold-sensitive dna mutant isolated in this lab by J. Couch and C. Greiner by using nitrosoguanidine mutagenesis and replica plating. This dna(Cs) gene was found to be cotransducible with ilv. We prepared phage P1

VOL. 133, 1978


strain BE280 (ilv-280 tna-4 phoS3) and infected both PC5 and CJ2 with this phage stock. Heat-resistant colonies (those able to form colonies at 4100) were selected from phage P1infected PC5 cells, and cold-resistant colonies (those able to form colonies at 2000) were selected from Pl-infected CJ2 cells. These selected colonies were purified and assayed for tna and phoS phenotypes. A large number of cold-resistant transductants of CJ2 can be isolated, but large numbers of heat-resistant transductants of PC5 at 410C were never obtained. It was then decided to grow P1 phage on PC5 and do the reciprocal transduction. First ilv+ transductants were isolated from BE280 cells infected with phage P1 grown on PC5. These ilv+ transductants selected at 320C were purified and scored for thermosensitivity and for tna and phoS phenotypes. The results of these transduction experiments are presented in Table 2. The dna markers carried by PC5 and CJ2 are both cotransducible with ilv at a low frequency (about 5%) and with tna and phoS at high frequencies of 85 and 65%, respectively. These data suggest that the order of these genes is: dnaA-tna-phoS-ilv. The results of further analysis of the genotypes of the transductants are summarized in Table 3. The absence of transductants having the Cr (cold-resistant) tna' phosS genotype from crosses 1 and 2, which is not likely due to its lethality, eliminated the possibility of the order tnaA-dnaA-phoS-ilv, as it appears on the E. coli map published in 1972 (32). The results from crosses 3 and 4 also favor the order with dnaA and phoS at different sides of tna. We also selected 40 additional temperature-sensitive ilv+ transductants from the cross grown on


identical to crosses 3 and 4. All of these 40 temperature-sensitive, ilv+ transductants were found to be tna' and phoS+, further strengthening the proposed gene order. It is apparent that both the dnaA5 and dnaA(Cs) carried by CJ2 map at similar, if not identical, positions. Construction of double mutants. E. coli FK8133 is a K-12 strain derived from LW126, a dnaC(Cs) mutant (34). Strain FK8133 was constructed first by introducing ilvD88 (from AB2279) into LW126 via P1 transduction. Then the dnaA5 (from PC5) marker was put into the resulting ilv auxotroph, FK1263, via P1 transduction by selecting ilv+ transductants. Heat and cold sensitivities of these transductants were tested by their ability to form colonies at 41 and 200C. Those colonies that can only grow at 320C, and not at 41 or 200C, were retested on rich LCTG plates. These double mutants were tested for temperature sensitivity of DNA synthesis by the double labeling technique. The dnaA gene product acts before that ofdnaC The presumptive double mutants were inoculated into the labeling medium and incubated at 320C without shaking. The overnight culture was inoculated into fresh labeling medium and incubated at 320C with shaking. After the cells were growing exponentially (usually in 2 h), subcultures were transferred to 20 and 410C at 0 min. From control experiments we found that after 2.5 h at 410C or 6.5 h at 200C, DNA synthesis stops and the cells are still viable if they are transferred back to 32°C. As a consequence, the experiment was designed so that the subculture at 410C was divided into three parts at 150 min: one was transferred to 200C, another was transferred to 320C, and the third remained at 410C. At 6.5 h, the original subcul-

TABLE 2. Transduction frequenciesa Donor

Cross Recipient




BE280 ( ilv-280 tna-4phoS3)

Selected marker Cr

Unselected marker

ilv-280 tna-4

phoS3 2




Cotransduction frequency (%)

19/483 81/95 72/95

3.9 85 76





154/179 107/179

86 60






HS tna+ phoS+

6/114 65/114 103/114

5.3 57 90



PC5 (dnaA5)


HE; tna+

7/139 86/139 118/139

5.0 62 85

phoS' a

Transductants with unselected markers/total transductant

Designations: Cr, cold resistant; HT, heat sensitive.




TABLE 3. Genotypes of transductantsa Cross





Cr tna+ phoSi Cr tna-4phoSg Cr tna-4phoS3 Cr tna+ phoS3

14/95 23/95 58/95 0/95

14.7 24.3 61.0 0


Cr tna+ phoSi

Cr tna-4phoSi Cr tna-4 phoS3 Cr tna+ phoS3

25/179 47/179 127/179 0/179

14.0 26.3 59.7 0

phoS3 tna-4 Hr phoS+ tna-4 Hr phoS tna+ Hr phoS+ tna+ H5

65/114 32/114 11/114 6/114

57.0 28.1 9.7 5.2


61.9 86/139 phoS3 tna-4 Hr 23.0 32/139 phoS' tna-4 Hr 10.1 14/139 phoSg tna+ Hr 5.0 7/139 phoS' tna+ Hs a Designations: Cr, cold resistant; H8, heat sensitive;


Hr, heat resistant. b S/T, number of transductants carrying the designated genotype per total number of transductants.

ture at 200C was similarly divided with one part transferred to 410C one transferred to 320C, and one part kept Et 200C. A 50-1l portion of each culture was wil hdrawn for the determination of protein andl DNA synthesis regularly during the course of the entire experiment. Figure 1 shows the results of the experiment with this double mutant FK8133 and the strains from which the dna markers were derived, PC5 and LW126. The left column of Fig. 1 (a, c, e) shows the results of the three strains first transferred to 200C and then to 410C. The curves in the right column show the cultures treated with the reverse order of shifting between restrictive temperatures. After being transferred to 200C, DNA synthesis in FK8133 (Fig. la) stopped gradually, as it did in LW126 (Fig. lc). The subculture of the original culture at 200C transferred to 320C at 6.5 h resumed DNA synthesis almost immediately, indicating that the cells are still viable after their exposure to the restrictive temperature. No further DNA synthesis, also similar to the behavior of LW126, could be detected in the subculture remaining at 200C. The subculture shifted from 20 to 410C (Fig. la) showed a 35% increase of DNA content, much less than that shown by the subculture shifted from 20 to 320C. This indicates either that FK8133 is not thermosensitive in DNA synthesis or that the dnaA gene product acts before that of dnaC. This is because during the first 200C treatment, the cells can complete the step con-

trolled by the dnaA gene in the DNA reinitiation process. In the subsequent 410C treatment, no more active dnaA gene product is produced, but the denatured dnaC gene product can be renatured or replenished by de novo synthesis at 410C. Under this condition, a new round of DNA replication would be expected to be completed under the assumption that these two genes act independently with dnaA preceding dnaC. Figure lc shows that the dnaC(Cs) mutation carried by LW126 only affected DNA synthesis at 200C, not at 32 or 410C. In Fig. le DNA synthesis in PC5, a dnaA(Ts) mutant, stopped very quickly after the culture was transferred from 20 to 410C after 6.5 h of incubation at the lower temperature. This "quick-stop" phenotype may have been due to the extremely slow growth rate of PC5 at 200C (the estimated doubling time is 3.5 h), and the residue DNA synthesis became less noticeable. It is apparent from Fig. le and f that 410C, not 20 or 320C, is the restrictive temperature for DNA synthesis in dnaA5 mutants. The thermosensitivity in DNA synthesis of FK8133 is clearly shown in Fig. lb. DNA synthesis stopped gradually at 410C, and no further synthesis could be detected in the 410C culture or the culture shifted to 200C at 150 min. The cells were still viable at the time of transfer to 200C because DNA synthesis resumed in the subculture transferred to 320C. These results rule out the possibility that FK8133 is not thermosensitive in DNA replication and confirmed our previous assertion that the dnaA gene product acts before that of dnaC. Each of the dna markers in FK8133 affected DNA synthesis at one restrictive temperature only, and 6.5 h of incubation at 200C did not affect the viability of single and double mutants (Fig. ld and e). That protein synthesis proceeded at different non-zero rates in all subcultures under all conditions tested indicated that all three strains are dna mutants. Some properties of the double mutant FK8133. The doubling time of FK8133 in minimal medium at 320C was about 140 min. Also, the cell lengths in an exponentially growing population were quite heterogeneous. Most of the cells form filaments at the permissive temperature. The filaments elongate at restrictive temperatures (20 and 4100) and can usually reach the length of 10 to 20 normal cells. The cells grew faster in minimal medium supplemented with 1% Casamino Acids (doubling time is about 80 min) than in L-broth (about 100 min). The reversion frequencies of FK8133 to cold resistance and heat resistance were tested by growing four cultures of newly cloned cells to stationary phase, spreading them on LCTG plates, and then incubating them at 20 or 410C.


E 1000 500r

(d) LW126

(c) LW126

'137 100 50












2 4 6 8 10



2 4 6

8 10

Time, hours FIG. 1. DNA and protein synthesis in temperature-sensitive and cold-sensitive E. coli mutants. (a) and (b), FK8133 [dnaA(Ts) and dnaC(Cs)]; (c) and (d), LW126 [dnaC(Cs)J; (e) and (D), PC5 [dnaA(Ts)]. ,*, and A represent protein content of cells. O, 0, and A represent DNA content of cells.

Temperature histories and notation

410C (0,O)

a, c, and e

32°C (A,A)

320C 200C (E,O)

6.5 h


b, d, and f

10 h

410C (-,0) 320C

320C (A,A)

200C (E,E) 2.5 h 0h Arrows on graphs indicate times of temperature shifts. 759

10 h



The reversion frequency of thermosensitvity was (3.3 ± 1.0) x 10- and (2.2 ± 1.0) x 10-7 for cold sensitivity. These values indicate that each of the two dna mutations is a single mutation and that the two lesions in the same strain do not interfere with each other.

DISCUSSION Mapping of the dnaA gene. Hirota et al. (14) have proposed the order dnaA-tna-phoSilv. But the number of transductants they scored was too small to be statistically significant. Wehr et al. (36) mapped four cold-sensitive dnaA alleles and suggested that the dnaA(Cs) gene be located between tna and phoS. By using strain BE280 we have shown clearly that dnaA5 and the cold-sensitive dnaA(Cs) allele carried by CJ2 map very close together if not at the same site on the E. coli genetic map. This is supported by three sets of transduction linkage data (dnaA with ilv, tna, and phoS) and a pair of transduction and reciprocal transduction experiments. The absence of one class of transductant (Cr tna+ phoS; cr for cold resistance) in crosses 1 and 2 strengthens the evidence for the gene order of dnaA-tna-phoS-ilv. There is little reason to believe that this genotype is lethal. The sequential function of the dnaA and dnaC gene products. Before concerning ourselves with the function of the dnaA and dnaC gene products, we will first discuss the existence of their products. The dnaC gene product has been isolated, purified, and demonstrated in an in vitro DNA synthesis system (37). The dnaA gene product has not been isolated, but its in vivo function in DNA synthesis has been found to influence the synthesis of RNA during initiation by Messer and his co-workers (24). Since dnaA(Ts) was recently shown to be dominant and to act in trans in dnaA(Ts)/dnaA merogenotes (42), it is very likely that a dnaA gene product is synthesized. This gene product must be so regulated that it is synthesized or activated only at the beginning of each replication cycle. The question of the existence and function of the dnaA gene product can be approached in another way by obtaining a synchronizable dnaA(Ts) mutant and doing the same kind of experiments as we did for dnaC(Ts) in another paper (manuscript in preparation). For simplicity in the following discussions, we will assume that dnaA does have a gene product. By using the reciprocal shift method, i.e., by shifting a culture of a double mutant between two restrictive temperatures in both possible combinations, our results suggest that dnaA acts before dnaC in the process of initiation of DNA replication. FK8133 [dnaA5(Ts), dnaC(Cs)] no longer synthesized DNA after being shifted from


41 to 20°C but increased its DNA content by 35% after being transferred from 20 to 410C. If all cells finish on-going DNA replication and stop at the same place after completion of the dnaA function at 20°C, they should double their DNA content after sufficiently long incubation at 41°C. The reasons for the increase of only 35% may be the following. (i) The growth rate of FK8133 at 200C is very slow judging from the ['4C]leucine uptake. The culture was incubated at 200C for 6.5 h and then transferred to 410C. During the 6.5-h incubation at 20°C, a small fraction of the cell population may not have finished the step controlled by the dnaA gene product for the next initiation, thus reducing the possible yield of new DNA synthesis at 41°C. (ii) The synthesis of the dnaC gene product or its renaturation at 41°C may be slow, and it is possible that a higher yield of DNA synthesis would be obtained if the culture were allowed to remain longer at 410C. (iii) There may be some interaction between the gene product of dnaA and dnaC. (iv) The heat-inactivated gene products may already occupy some important site in the initiation machinery and thus prevent the newly synthesized product from functioning efficiently. (v) The renatured gene product may be less effective than the wild-type gene product. These are by no means all of the possible explanations, but the most important points are that the dnaA gene product acts before that of dnaC and that they may work independently in the process of initiation of chromosome replication. Comparing the DNA synthesis in FK8133 and PC5 in Fig. lb and f after the culture was shifted from 41 to 200C, we found that the DNA content in PC5 only increases about 50%, whereas that in FK8133 does not increase at all for 8 h. Figure lf indicates that after exposure to 410C, the resumption in DNA synthesis at 200C is much slower than that at 320C. We see the same phenomenon in Fig. ld. We did not detect any increase in DNA synthesis with FK8133 after shifting from 41 to 200C (Fig. lb) and are very confident in the sensitivity of the radioactivity measurement. From these results we suggest that dnaA acts before dnaC. Messer and his co-workers (24, 39) reported that dnaA, but not dnaC, may be involved in the synthesis of specific RNA, which is directly related to the initiation process of DNA replication. Zyskind et al. (40) very recently found that addition of rifampin to a culture of thermoreversible dnaA mutants at a restrictive temperature, causing 99% inhibition of RNA synthesis, will inhibit reinitiation after the culture is shifted to the permissive temperature, but not in dnaC(Ts) mutants. The rifampin


VOL. 133, 1978

inhibition of reinitiation is not observed when a rifampin-resistant RNA polymerase gene is introduced into the dnaA mutant. So it seems that RNA polymerase and the dnaA gene or gene product may be involved in a transcriptional event during the initiation process. By using nalidixic acid instead of rifampin, they found that dnaC may be responsible for the first deoxyribonucleotide polymerization event. They came to a conclusion similar to ours that dnaA may act before dnaC in the initiation process. By using the same rationale and constructing heat- and cold-sensitive phage mutants, Jarvik and Botstein (16) successfully studied the order of genetically controlled steps in the assembly of bacteriophage P22. Hartwell and his co-workers (10, 13) also explored the sequential gene function in the control of the cell cycle and genetically controlled the process of the initiation of DNA synthesis in Saccharomyces cerevisiae. In this paper we demonstrated the use of genetic techniques to solve problems concerning the initiation of DNA replication in E. coli. The same rationale can be used to construct other combinations of double dna mutants. Our method is carried out completely in vivo, does not involve the use of any antibiotics, and should also be useful for the in vitro study of DNA synthesis. ACKNOWLEDGMENTS We are grateful for the excellent technical assistance of Ruth Ford and Carol Greiner. This work was supported by Public Health Service grant GM-22021 from the National Institute of General Medical Sciences.

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32. 33.


35. 36.


dnaA acts before dnaC in the initiation of DNA replication.

JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 755-762 0021-9193/78/0133-075502.00/0 Copyright X 1978 American Society for Microbiology Vol. 133, No. 2 Print...
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