Heterogeneity in E. coli RecBCD Helicase-DNA Binding and Base Pair Melting

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RecBCD is a crucial enzyme in double-stranded DNA break repair in E. coli.
• It functions as a helicase/nuclease, initiating repair by binding DNA ends.
• The enzyme's ability to melt DNA base pairs is key to its function.

Purpose of the Study:

• To investigate the thermodynamic and structural properties of RecBCD-DNA initiation complexes.
• To understand how RecBCD interacts with DNA ends and melts base pairs.
• To elucidate the role of conformational dynamics in RecBCD function.

Main Methods:

• Thermodynamic measurements (enthalpy changes) of RecBCD binding to DNA with varying ssDNA tail lengths.
• Cryo-electron microscopy (Cryo-EM) to determine structures of RecBCD alone and bound to DNA.
• Analysis of conformational heterogeneities in RecBCD subunits (RecB nuclease domain and RecD).

Main Results:

• RecBCD interacts with single-stranded DNA tails up to 17-18 nucleotides.
• Upon binding blunt DNA ends, RecBCD melts at least 10-11 base pairs.
• DNA binding reduces conformational heterogeneity, stabilizing the RecB nuclease domain and RecD subunit.

Conclusions:

• RecBCD exhibits significant conformational flexibility, particularly in its RecB nuclease domain.
• DNA binding induces structural changes, stabilizing key domains and increasing DNA melting.
• The extent of DNA melting is variable and may regulate the initiation of helicase activity.