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Related Experiment Videos

Re-replication from non-sequesterable origins generates three-nucleoid cells which divide asymmetrically.

Trond Bach1, Kirsten Skarstad

  • 1Department of Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, 0310 Oslo, Norway.

Molecular Microbiology
|March 11, 2004
PubMed
Summary

Altering replication origins in Escherichia coli prevents sequestration, leading to extra DNA replication rounds. This results in premature cell division and the formation of additional daughter cells.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Cell Biology

Background:

  • Rapidly growing Escherichia coli cells initiate replication at multiple origins (oriCs) synchronously once per cell cycle.
  • Sequestration, involving SeqA protein and Dam methylation of GATC sites in oriC, prevents immediate re-initiation of origins.

Purpose of the Study:

  • To investigate the role of GATC sites in oriC in the sequestration mechanism.
  • To understand the consequences of bypassing sequestration on DNA replication and cell division in Escherichia coli.

Main Methods:

  • Genetic modification of GATC sites to GTTC within the oriC region of Escherichia coli.
  • Observation and analysis of replication initiation, nucleoid content, and cell division in mutant strains.
  • Comparison of mutant phenotypes with wild-type and SeqA-deficient cells.

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Main Results:

  • Mutating GATC sites to GTTC significantly reduced origin sequestration, similar to SeqA-deficient cells.
  • The mutant origins underwent re-initiation, confirming the requirement of GATC sites for sequestration.
  • Re-initiation led to cells with extra nucleoids, which divided into three daughter cells through sequential divisions.
  • Despite premature replication and division, the mutant exhibited relatively normal growth rates and cell size distribution.

Conclusions:

  • GATC sites within oriC are essential for the sequestration mechanism that ensures single-use of origins per cell cycle.
  • Bypassing sequestration triggers extra replication rounds, leading to premature cell division and increased daughter cell formation.
  • Escherichia coli possesses flexible cell cycle controls that can accommodate aberrant replication, segregation, and division events.