DNA damage alters binding conformations of E. coli single-stranded DNA-binding protein

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• Single-stranded DNA-binding proteins (SSBs) are crucial for DNA homeostasis, binding ssDNA to facilitate repair and replication.
• Escherichia coli SSB (EcSSB) is a well-studied homotetramer that binds ssDNA in distinct conformations (65-nt or 35-nt modes).

Purpose of the Study:

• To investigate the impact of DNA damage and structural alterations on EcSSB binding dynamics.
• To determine how EcSSB affinity, cooperativity, and conformation are affected by non-canonical DNA bases and non-DNA linkers.

Main Methods:

• Atomic force microscopy (AFM) was employed to visualize and quantify EcSSB binding to individual ssDNA molecules.
• Experimental constructs incorporated synthetic abasic sites and non-DNA linkers to mimic DNA damage and structural impediments.

Main Results:

• EcSSB binding affinity and conformation remained unchanged when one non-canonical nucleotide was present.
• Binding affinity for the 65-nt mode was significantly reduced by tandem abasic sites or a non-DNA spacer.
• Binding of two EcSSB tetramers in the 35-nt mode was preserved despite these structural alterations.

Conclusions:

• The presence of multiple DNA lesions, rather than single damaged sites, alters EcSSB's binding behavior.
• These findings provide insights into how accumulated environmental DNA damage may modulate genomic repair and replication processes.