Single-molecule studies of fork dynamics in Escherichia coli DNA replication

N. Tanner, S. Hamdan, S. Jergic, P. Schaeffer, N. Dixon, A. Oijen

Research output: Contribution to journalArticlepeer-review

Abstract

The process by which cells duplicate their genomes, DNA replication, is a precise process where accuracy and efficiency depend on specific protein-protein interactions between components of the replisome. In Escherichia coli, these key components include the DnaB helicase, DnaG primase, and DNA polymerase III (Pol III) (Fig. 1a). Since all nucleotide polymerases can synthesize polynucleotides in only the 5′ to 3′ direction, a universal feature of DNA replication is a molecular inequality in synthesis of the two strands of a chromosome. Thus, the leading strand is synthesized in a continuous manner, involving a DNA polymerase that remains constantly on the template; the other, lagging strand is synthesized in discrete segments, Okazaki fragments, involving a DNA polymerase with lower processivity but also requiring efficient cycling of components that repeatedly dissociate and reinitiate Okazaki fragments. In this article, the authors used single-molecule techniques to observe how the components of the replisome in E. coli regulate replication, specifically how the rate of leading strand synthesis is modulated so that it does not outpace lagging strand synthesis. By monitoring changes in the length of DNA during primer extension, where elongation means double-strand DNA formation and shortening means single-strand formation (DNA unwinding), the investigators determined polymerization kinetics as key components were added in an in vitro system (Fig. 1b). Results showed that DnaB-Pol III holoenzyme interaction is necessary to increase processivity of DNA synthesis as DnaB promotes dsDNA unwinding and fork propagation. Binding of DnaG decreases leading strand synthesis, thereby acting as a molecular brake to modulate processivity on the leading strand template. Moreover, interaction of a trimer of DnaG and DnaB terminates leading strand synthesis, suggesting that DnaG destabilizes the replisome, again consistent with its function as a molecular brake. The association is cooperative and shows that termination of replication is solely due to protein-protein interaction and not dependent on primase activity. This study thus shows how leading strand synthesis is modulated so that it does not outpace lagging strand replication, which has a slower rate of primer synthesis.

Original languageEnglish (US)
Pages (from-to)85-86
Number of pages2
JournalChemtracts
Volume21
Issue number3
StatePublished - Mar 2008
Externally publishedYes

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry
  • Molecular Biology

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