Recognition of DNA structure by 434 repressor

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• The bacteriophage 434 repressor binds to specific DNA sequences.
• The central bases of the 434 repressor binding site are not directly contacted by the protein.
• Previous studies suggested DNA overtwisting influences repressor-DNA binding affinity.

Purpose of the Study:

• To investigate the role of DNA twisting in bacteriophage 434 repressor binding.
• To determine how central base sequence variants affect binding affinity as a function of DNA twist.
• To elucidate the contribution of the N-terminal dimer interface to repressor-DNA interactions.

Main Methods:

• Assessing the binding affinity of central sequence variant 434 binding sites for 434 repressor.
• Measuring binding affinity as a function of the binding site's average twist.
• Analyzing repressor binding to DNA sites with insertions or deletions in the central region.
• Mutational analysis of the N-terminal dimer interface.

Main Results:

• Bacteriophage 434 repressor demonstrated a preference for relatively overwound binding sites over underwound forms.
• Maximal affinity enhancement occurred with a binding site that was initially underwound and resistant to twisting.
• The repressor could bind to a 15-base pair site with a central insertion but bound poorly to 12- and 13-base pair sites with central deletions.
• Mutations in the N-terminal dimer interface abolished central base discrimination and altered site size preferences.

Conclusions:

• DNA overtwisting is a key factor governing bacteriophage 434 repressor binding affinity.
• The N-terminal dimer interface is critical for determining central base preferences and site size selection.
• Repressor-DNA interactions involve complex structural changes, including DNA overtwisting, influenced by protein interfaces and DNA sequence.