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A novel cytoplasmic hemimethylated oriC binding activity

J Garwood1, M Kohiyama

  • 1Biochemical Genetics Group, Institut Jacques Monod, Université Paris, France.

The Journal of Biological Chemistry
|March 29, 1996
PubMed
Summary

Researchers discovered a novel protein binding activity specific to hemimethylated DNA in Escherichia coli. This activity, found in the cytoplasm, may play a role in DNA replication origin sequestration.

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Area of Science:

  • * Molecular Biology
  • * Microbiology
  • * Genetics

Background:

  • * DNA replication initiation in Escherichia coli is a complex process involving specific regulatory mechanisms.
  • * Hemimethylated DNA, arising after replication, plays a crucial role in cell cycle control and DNA segregation.
  • * Previous studies have identified several proteins involved in oriC regulation, but the complete picture remains elusive.

Purpose of the Study:

  • * To identify and characterize novel protein factors involved in the recognition and binding of hemimethylated DNA at the Escherichia coli replication origin (oriC).
  • * To investigate the cellular localization and purification of this novel binding activity.
  • * To determine the specific binding sites within oriC and explore its potential role in DNA replication regulation.

Main Methods:

  • * Used hemimethylated, fully methylated, and unmethylated oligonucleotide probes of the oriC region (+81-136) for DNA binding assays.
  • * Employed DNase footprinting analysis to precisely map protein-DNA interactions.
  • * Performed partial protein purification and analyzed binding activity in different cellular fractions and in seqA deletion mutants.
  • * Investigated the influence of cell cycle stage on binding activity in synchronized cultures.

Main Results:

  • * Characterized a novel hemimethylated DNA-specific protein binding activity localized in the cytoplasm.
  • * This activity preferentially protected hemimethylated GATC sites adjacent to the DnaA binding box (R1) and overlapping the integration host factor (IHF) binding site within oriC.
  • * The binding activity was reduced threefold in seqA deletion mutants, suggesting a potential link to SeqA function.
  • * Binding activity levels were not significantly affected by the cell cycle stage.

Conclusions:

  • * A novel hemimethylated DNA-binding protein activity involved in oriC regulation has been identified.
  • * This activity specifically targets key regulatory sites within the minimal oriC, suggesting a role in controlling replication initiation.
  • * The findings suggest a potential role for this activity in the sequestration of newly replicated, hemimethylated DNA origins, contributing to cell cycle progression.

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