Rates of DNA chain growth in Escherichia coli

Haim Manor, Murray P. Deutscher, Uriel Z. Littauer

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21 Scopus citations

Abstract

The thymidylic acid step-time, σT, was measured in two strains of Escherichia coli in order to obtain an estimate of the rate of DNA chain growth. (σT is defined as the average time required for adding the next deoxyribonucleotide to the end of a nascent DNA chain carrying thymidylic acid at its growing end.) For these measurements E. coli cultures were pulse-labeled with [3H]thymidine for 6, 10, 14 and 18 seconds. DNA was extracted from each sample and hydrolyzed enzymically to yield deoxyribonucleoside-3′-monophosphates from interior residues and dexoyribonucleosides from 3′-OH termini. Determination of the radioactivities in thymidine-3′-monophosphate and in thymidine showed that the ratio [3H]thymidine [3H]thymidine + [3H]thymidine-3′-monophosphate decreased continuously during the labeling period. Such a decrease is to be expected if deoxyribonucleoside-5′-phosphates are utilized as precursors of chromosome replication in vivo and if DNA chains grow in the direction 5′ → 3′. These data allowed a calculation of σT. In E. coli BT- (thymine-) grown at 18.5, 20 and 25 °C in a minimal medium supplemented with glucose and 0.3 to 0.4 μg thymine/ml., σT was found to be 20 to 23, 13 to 15 and 6 to 9 msec, respectively. The corresponding rates of DNA chain growth were estimated to be 43 to 50, 67 to 77 and 111 to 167 nucleotides per second. In these cultures the turbidity increased exponentially but DNA synthesis was partially inhibited by the low level of thymine in the medium. In E. coli B grown at 20.5 †C in minimal medium supplemented with glucose, turbidity and DNA content both increased exponentially. In this case σT was found to be 4 to 7 msec and the rate of DNA chain growth was estimated to be 143 to 250 nucleotides per second. This estimate of the rate of chain growth is somewhat slower than the over-all rate of movement of the replication fork which was reckoned to be 300 to 420 nucleotides per second for E. coli B grown at 20.5 °C. It is, however, 11 to 19 times faster than the rate of RNA chain growth.

Original languageEnglish (US)
Pages (from-to)503-524
Number of pages22
JournalJournal of molecular biology
Volume61
Issue number3
DOIs
StatePublished - Nov 14 1971

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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