Analysis of the 5′-termini of nascent DNA chains synthesized in permeable cells of Bacillus subtilis

Gaspar Banfalvi, Nilima Sarkar

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18 Citations (Scopus)

Abstract

The nascent DNA synthesized by permeable cells of Bacillus subtilis in the presence of 5′-mercurideoxycytidine triphosphate and 2′,3′-dideoxyATP has been isolated and characterized. The newly synthesized DNA was isolated free from other cellular nucleic acids by affinity chromatography on thiol-substituted agarose. The number average chain length of the nascent DNA synthesized in one minute at 25 °C was 33 nucleotide residues, due to the chain-terminating action of 2′,3′-dideoxyATP. Several lines of evidence indicated that at least 90% of the DNA thus isolated carried a terminally phosphorylated RNA moiety at its 5′-end: (1) the nascent DNA was resistant to exonucleolytic degradation by spleen phosphodiesterase unless first hydrolyzed by strong alkali or ribonuclease; (2) the 5′-termini of nascent DNA could not be phosphorylated by polynucleotide kinase unless first treated with alkaline phosphatase or subjected to hydrolysis by strong alkali or ribonuclease; (3) alkaline hydrolysis of nascent DNA labeled with 32P at the 5′-end released unlabeled DNA with a free 5′-terminus and 32P-labeled ribonucleoside 3′,5′-bisphosphates; (4) ribonuclease degradation of similarly labeled material produced an unlabeled DNA-containing polynucleotide fraction and 32P-labeled ribo-oligonucleotides; (5) chromatography on dihydroxyboryl cellulose showed that the RNA moiety lacked a 3′-terminal cis-diol grouping (even after treatment with alkaline phosphatase) unless first subjected to the 3′-exonucleolytic action of bacteriophage T4 DNA polymerase. The sequence of the ribonucleotide chains was elucidated by end-group labeling with polynucleotide kinase and digestion with various ribonucleases. The ribonucleotide moiety was primarily three and four residues in length with the predominant sequence (pp)pApG(pC)1-2pDNA. The possibility that it represents a primer for discontinuous DNA synthesis is discussed.

Original languageEnglish
Pages (from-to)147-169
Number of pages23
JournalJournal of molecular biology
Volume163
Issue number2
DOIs
Publication statusPublished - Jan 15 1983

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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