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Kinetic analysis of DNA compaction by mycobacterial integration host factor at the single-molecule level

Yuanyuan Chen1, Zhengyan Zhan2, Hongtai Zhang3

  • 1National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100049, China; Core Facility for Protein Research, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

Tuberculosis (Edinburgh, Scotland)
|November 17, 2019
PubMed

Insights

Mycobacterial integration host factor (mIHF) is a nucleoid-associated protein that compacts DNA. This study reveals how M. smegmatis IHF (MsIHF) condenses DNA through a three-step mechanism, aiding mycobacterial chromosome organization.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Biophysics

Background:

  • Nucleoid-associated proteins (NAPs) are crucial for bacterial chromosome condensation and organization.
  • Mycobacterial integration host factor (mIHF) is a key NAP in mycobacteria, influencing DNA integration and structure.
  • Understanding mIHF's function is vital for deciphering mycobacterial DNA organization mechanisms.

Purpose of the Study:

  • To investigate the DNA-binding and compaction properties of M. smegmatis IHF (MsIHF).
  • To elucidate the mechanism of DNA condensation mediated by MsIHF at the single-molecule level.

Main Methods:

  • Bio-layer interferometry (BLI) to assess DNA binding and ion strength sensitivity.
  • Total internal reflection fluorescence microscopy (TIRFM) for single-molecule DNA compaction visualization.
  • Atomic force microscopy (AFM) for observing higher-order DNA structures.

Main Results:

  • MsIHF binds 30-bp dsDNA fragments independently of sequence and is sensitive to ion strength.
  • MsIHF efficiently compacts λ DNA at concentrations of 0.25 and 1.0 μM, achieving packing ratios >10.
  • Kinetic analysis identified a three-step DNA compaction mechanism involving two compaction phases and a lag phase.

Conclusions:

  • MsIHF plays a significant role in DNA condensation within mycobacteria.
  • The findings provide insights into the step-wise mechanism of DNA compaction by MsIHF.
  • This research contributes to a deeper understanding of chromosomal organization in mycobacteria.
Keywords:
DNA compactionMycobacterial integration host factor (mIHF)Nucleoid-associated proteinsSingle-molecule

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