DNA Synthesis in Nucleotide Permeable Escherichia coli Cells

Abstract

Cells made permeable to nucleotides by treatment with ether synthesize DNA from exogenous deoxynucleoside triphosphates in a semi-conservative fashion. The reaction proceeds from sites of previous synthesis on the chromosome, and not detectably from new sites. Short chains appear as precursors of long units and have a mean sedimentation coefficient of about 10 S in alkali, like Okazaki-pieces when synthesized in the presence of 10 to 20p.M deoxynucleoside triphosphates, and of only 5 S when synthesized in the presence of 1 p.M dNTP. Under the former conditions short chains contain not more than half of the newly synthesized DNA; under the latter conditions they can contain more than half, the remainder of the newly synthesized DNA being in long units. Nascent short chains labelled after several minutes of synthesis in the presence of 5-bromodeoxyuridine 5'-triphosphate at high deoxynucleoside triphosphate concentrations show bimodal density distribution in an alkaline Cs,SO, equilibrium gradient with peaks of material corresponding to the density of heavy single strands and almost light single strands, respectively. The implications of these findings are discussed. Judged from controls, repair synthesis was not a source of error in these experiments.

DNA Synthesis in Nucleotide-Permeable Escherichia coli Cells. The Effects of Nucleotide Analogues on DNA Synthesis

“…The alkali buffers (pH 12.8) consisted of 0.05 M glycine, 0.15 M NaOH and 1 mM EDTA; sucrose gradients additionally contained 0.5 M NaC1. Further details have been described [3]. I n the profiles o f this paper the direction of sedimentation and increase in density is from right to left.…”

“…The specific activity was more than 25 Ci Gradients Alkaline and neutral sucrose (5 -20 o/o) velocity sedimentation was performed in the Beckman SW27 rotor for 15 h (20 "C) a t 26000 and 22000 rev./min, respectively, as described previously [3]. Alkaline cesium sulphate gradients (average density 1.46 g/ml) were performed in a Beckman fixed-angle rotor 50 Ti for 60 h (20 "C) a t 32000 rev./min.…”

“…After a few minutes of incorporation a t 25 "C, DNA synthesis was stopped with cold Ksucrose medium, followed by centrifugation. The cells were lysed with lysozyme and sarcosyl in the presence of RNAase and pronase as described previously [3].…”

“…The synthesis of Escherichia coli DNA is semiconservative and proceeds from sites of previous synthesis on the chromosome [3]. At least one half of a pulse label is first in short segments (Okazaki pieces) which can be integrated into larger DNA [3].…”

“…The synthesis of Escherichia coli DNA is semiconservative and proceeds from sites of previous synthesis on the chromosome [3]. At least one half of a pulse label is first in short segments (Okazaki pieces) which can be integrated into larger DNA [3]. NMN, which can discharge the adenylated intermediate form of DNA ligase [4], drastically reduced the size of newly synthesized E. coli DNA chains, although NAD failed to stimulate DNA synthesis as Denhardt and Burgess [5] reported for crude extracts from cells infected with phage pX174.…”