Distinct double- and single-stranded DNA binding of E. coli replicative DNA polymerase III alpha subunit

Area of Science:

• Molecular Biology
• Biophysics
• Biochemistry

Background:

• The alpha subunit is the active polymerase in the 10-subunit bacterial replicase of E. coli.
• The C-terminal region of the alpha subunit is predicted to contain an oligonucleotide binding (OB-fold) domain.

Purpose of the Study:

• To investigate the DNA binding properties of the alpha subunit of E. coli DNA polymerase III.
• To determine the specific DNA binding domains and their affinities for double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA).

Main Methods:

• Utilized optical tweezers experiments to probe protein-DNA interactions.
• Performed DNA stretching measurements to determine equilibrium association constants for alpha subunit and its fragments binding to dsDNA and ssDNA.

Main Results:

• The alpha subunit exhibits distinct binding affinities for both dsDNA and ssDNA through separate subdomains.
• Binding to dsDNA stabilizes the DNA helix, hindering melting and reannealing.
• ssDNA binding is localized to the C-terminal OB-fold domain.
• A tandem helix-hairpin-helix (HhH)2 motif significantly contributes to dsDNA binding.

Conclusions:

• The alpha subunit possesses dual DNA-binding capabilities mediated by distinct structural motifs.
• The OB-fold domain is responsible for ssDNA binding, while the HhH motif mediates dsDNA binding.
• These interactions play a crucial role in the function and regulation of bacterial DNA replication.