Enhancer-origin interaction in plasmid R6K involves a DNA loop mediated by initiator protein

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Plasmid R6K DNA replication initiation requires specific origin sequences.
• The ori beta sequence alone is insufficient for replication initiation.

Purpose of the Study:

• To investigate the mechanism of initiator protein binding and DNA looping in R6K plasmid replication.
• To elucidate the role of the ori gamma sequence in facilitating ori beta function.

Main Methods:

• Cyclization enhancement assay
• Exonuclease III protection assay
• Electron microscopy
• Chemical footprinting

Main Results:

• Initiator protein binding to ori gamma enhances binding at ori beta through long-range cooperativity.
• Protein-mediated interaction between ori gamma and ori beta forms a DNA loop.
• Evidence of DNA looping confirmed by multiple biochemical and biophysical techniques.

Conclusions:

• DNA looping mediated by initiator protein binding is a crucial early step in R6K plasmid DNA replication initiation at ori beta.
• The ori gamma sequence acts in cis to facilitate replication initiation at ori beta via protein-mediated DNA looping.