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Related Experiment Videos

Fis cannot support oriC DNA replication in vitro

H Hiasa1, K J Marians

  • 1Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.

The Journal of Biological Chemistry
|October 7, 1994
PubMed
Summary

Histone-like proteins HU and IHF stimulate DNA replication at oriC, while Fis inhibits it. Fis

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Biochemistry

Background:

  • Histone-like proteins play crucial roles in DNA organization and replication in bacteria.
  • HU, IHF, and Fis are key nucleoid-associated proteins influencing DNA processes.
  • Understanding their specific roles in DNA replication is essential for bacterial genetics.

Purpose of the Study:

  • To investigate the in vitro effects of histone-like proteins HU, IHF, and Fis on DNA replication using oriC and pBR322 systems.
  • To determine the influence of these proteins on DNA unwinding at replication origins.
  • To clarify the role of Fis in oriC DNA replication.

Main Methods:

  • In vitro DNA replication assays using oriC and pBR322 DNA substrates.
  • Addition of varying concentrations of HU, IHF, and Fis proteins.
  • Analysis of DNA unwinding at replication origins.
  • Assessment of DNA replication levels in response to protein additions.

Main Results:

  • Low concentrations of HU and IHF stimulated oriC DNA replication.
  • High concentrations of Fis inhibited oriC DNA replication, an effect counteracted by HU, IHF, or RNA polymerase.
  • DNA unwinding at the origin correlated with increased DNA replication in the oriC system.
  • In the pBR322 system, these proteins only caused inhibition, not stimulation.

Conclusions:

  • The stimulatory effect of HU and IHF on oriC DNA replication is concentration-dependent.
  • Fis exhibits inhibitory effects on oriC DNA replication, which can be modulated by other proteins.
  • The in vivo dependence of oriC DNA replication on Fis is not solely due to enhanced localized DNA unwinding.

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