A New Element in the Control of DNA Initiation in Escherichia coli

Abstract

When a thermosensitive mutant of E. coli affected in DNA initiation is heated to-41°, a protein needed for DNA initiation is irreversibly denatured. When these bacteria are incubated at 300 in the presence of several concentrations of chloramphenicol, DNA synthesis is greatly stimulated at a chloramphenicol concentration of 2.5 gg/ml, and then sharply decreases. This stimulation of DNA synthesis appears to be caused by an increased frequency of initiation. The existence of an "anti-initiator" protein controlling the frequency of chromosomal initiation is proposed.Chromosomes replicate in Escherichia coli by DNA initiation and DNA elongation. Protein synthesis is needed for the first process but not for the second (1), and there is evidence that two different proteins are required for the initiation process (2, 3).The isolation of several thermosensitive mutants of E. coli defective in DNA synthesis suggests that many gene functions are involved in this process (4, 5). dnaA and dnaC are affected in DNA initiation (6, 7). However, no correlation has yet been established between the two proteins normally required for DNA initiation and the thermosensitive lesions of dnaA and dnaC mutants. In a dnaA mutant, a protein required for DNA initiation is irreversibly denatured at 410 and rounds of chromosome replication already in progress can be completed, but new rounds cannot be initiated (6).The purpose of this work is to study in more detail the characteristics of the initiation process altered in a dnaA mutant. MATERIALS AND METHODSA derivative of E. coli K12, CRT 46 was used that has the following characteristics: F-Thr-Leu-Thy-Br-Ilv-lac yMal-T46SXr. . Further manipulations were as described above. Density-Shift Experiments. A culture of CRT46 was grown overnight at 300 in M63 medium supplemented as described above and with 5 uCi of/ ['4C]thymine; 60 ,ug of thymine per ml. After 16 hr, the culture was diluted 10-fold (up to 10 ml) with the same medium. When a cell density of 2 X 10' bacteria per ml was attained, the culture was incubated at 410 for 1 hr, rapidly cooled, and centrifuged. The pellet was washed once with cold M63 medium minus ammonium sulfate, suspended in 10 ml of medium 63 supplemented as described above, with the exception that NH4C1 and glucose has been replaced by 2 mg each of 1"NH4Cl (97.2 atom % 15N; Isomet Corp.) and [2H ]glucose (98.2 atom % 2H; Volk Radiochemical Co.) per ml. The culture was divided in halves; chloramphenicol (2.5,Mg/ml) was added to one of them, and both were incubated at 30°. 5 min later, ['H thymidine (10 ,uCi/,ug per ml) was added, and 2.5-ml aliquots were withdrawn at 40 and 90 min. Cultures were centrifuged, and pellets were suspended in 1 ml of a solution containing 10 mM TrissHCl-10 mM ethylenediamine tetraacetate (EDTA) (pH 8.5)-0.5% sodium dodecyl sulfate-2 mg of Pronase per ml. After 10 min at 450, the lysates were incubated 2 hr more at 370, and centrifuged at 20,000 X g for 10 min; the supernatants were recovered. The 40-min samples were diluted with 1.9...

Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus

“…DNA synthesis in wild-type cells (RN4220), as determined by incorporation of [ 3 H]thymidine into acidinsoluble fractions, was completely blocked within 10 min after addition of 100 lg/ml nor¯oxacin, whereas 30 min after the addition of 100 lg/ml chloramphenicol DNA synthesis had decreased by only 20%. Nor¯oxacin acts on DNA topoisomerases to prevent progression of the replication fork, whereas chloramphenicol inhibits the initiation of DNA replication (Blau and Mordoh 1972). In TS0605 and TS0046 30 min after shifting the temperature to 43°C rates of DNA replication remained approximately 100% and 50%, respectively, of those prior to the shift.…”

Isolation and characterization of temperature-sensitive mutants of the Staphylococcus aureus dnaC gene

“…4). The former inhibits the swivelase activity of DNA gyrase, which is required for progression of the replication fork, whereas chloramphenicol inhibits protein synthesis leading to an inhibition of the initiation of DNA replication [15]. The rate of DNA synthesis in TS0021, TS0052, TS0685, and TS0686 decreased to less than 30% within 15 min after the increase in temperature, indicating faststop phenotypes for DNA synthesis in these mutants (Fig.…”

Copy-number mutants of the plasmid carrying the replication origin of the Escherichia coli chromosome: evidence for a control region of replication.

“…On the other hand, the existence of a negatively acting protein(s) for initiation of chromosomal replication has also been suggested (34,35).…”