A second high affinity HU binding site in the phage Mu transpososome

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Bacteriophage Mu transposition involves nucleoprotein intermediates called transpososomes.
• Escherichia coli HU protein, a sequence-independent DNA binder, is crucial for transpososome assembly.
• HU protein exhibits significantly higher affinity for Mu transpososomes than for supercoiled DNA.

Purpose of the Study:

• To identify the specific binding site of HU protein on Mu transpososomes.
• To elucidate the mechanism of high-affinity HU binding to Mu transposition complexes.
• To investigate the role of protein-protein interactions versus DNA structure in HU binding.

Main Methods:

• Immunoelectron microscopy to visualize HU binding sites.
• Chemical modification of Mu A protein to assess protein-protein interactions.
• Salt washes to remove and reassemble HU protein.
• Functional assays using eukaryotic HMG-1 protein.

Main Results:

• High-affinity HU binding occurs at a region distinct from the previously identified Mu left end site.
• HU protein can be efficiently removed and reassembled into the transpososome complex.
• Chemical modification of Mu A protein does not prevent HU reassembly, indicating A-HU interactions are not primary.
• Eukaryotic HMG-1 protein can functionally substitute for HU in transpososome formation.

Conclusions:

• HU protein binds to Mu transpososomes at a site separate from the Mu left end.
• High-affinity HU binding is mediated by recognition of an altered DNA structure within the transpososome.
• Protein-protein interactions with Mu A are unlikely to be the primary driver of HU binding.