Binding of E. coli single-stranded DNA binding protein to damaged DNA substrates measured at single molecule level using AFM.

Abstract

Single-stranded DNA binding proteins (SSBs) are essential cellular components, binding to transiently exposed regions of single-stranded DNA (ssDNA) with high affinity and sequence non-specificity to coordinate DNA repair and replication. E. coli SSB (Ec SSB), a homotetramer that wraps variable lengths of ssDNA in multiple conformations, is a model protein system with resolved structures that is well-studied across experimental conditions (temperature, salt, pH, substrate length, etc.). We use atomic force microscopy (AFM) to investigate the binding of SSB to individual ssDNA molecules. Furthermore, we introduce noncanonical DNA units that mimic naturally occurring DNA damage (synthetic abasic sites and non-DNA linker) into our experimental constructs at sites predicted to interact with binding Ec SSB. By measuring the fraction of DNA constructs with SSB bound as well as the number of SSB proteins bound per construct, we determine the protein binding affinity and cooperativity. We find that both the affinity and cooperativity of protein binding are affected by DNA damage. Changes in the binding and cooperative behaviors of SSB proteins across these constructs can inform how genomic repair and replication processes may change as environmental damage accumulates.