Origin of replication of the Bacillus subtilis chromosome: In vitro approach to the isolation of early replicating segments

M. Sasvári, G. Bánfalvi, K. F. Bott, N. Sarkar

Research output: Contribution to journalArticle

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Abstract

We have developed a permeable cell system for the study of the molecular mechanisms involved in the control and initiation of DNA replication at the origin of the Bacillus subtilis chromosome. Our system takes advantage of the synchronous initiation of DNA replication that occurs in outgrowing B. subtilis spores and the curtailment of DNA elongation by novobiocin. Early replicating DNA sequences were identified by the use of 5-mercury-dCTP as substrate, which allows the isolation of nascent DNA chains by affinity chromatography on thiol agarose. The average size of the isolated nascent DNA was 1,000 bp, and more than 80% of the nascent DNA chains had RNA primers at their 5' end. The study of the temporal order of chromosome replication near the origin using this experimental system showed that a segment containing recF and gyrB replicated earlier than a segment containing gyrA and part of the rRNA operon (rrnO). This observation is in agreement with previous in vivo data on the replication of origin region and supports the conclusion that the major activity in our in vitro system was the faithful replication of the ori region.

Original languageEnglish
Pages (from-to)1049-1055
Number of pages7
JournalDNA and Cell Biology
Volume14
Issue number12
Publication statusPublished - 1995

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Replication Origin
Bacillus subtilis
Chromosomes
DNA
DNA Replication
rRNA Operon
Novobiocin
Spores
Mercury
Affinity Chromatography
Sulfhydryl Compounds
Sepharose
In Vitro Techniques

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

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Origin of replication of the Bacillus subtilis chromosome : In vitro approach to the isolation of early replicating segments. / Sasvári, M.; Bánfalvi, G.; Bott, K. F.; Sarkar, N.

In: DNA and Cell Biology, Vol. 14, No. 12, 1995, p. 1049-1055.

Research output: Contribution to journalArticle

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