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

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 replica-tion. Escherichia coli SSB (EcSSB) is a homotetramer that wraps variable lengths of ssDNA in multiple conformations (typically occupying either 65 or 35 nt), which is well studied across experimental conditions of substrate length, salt, pH, temperature, etc. In this work, we use atomic force microscopy to investigate the binding of SSB to individual ssDNA molecules. We introduce non-canonical DNA bases that mimic naturally occurring DNA damage, synthetic abasic sites, as well as a non-DNA linker into our experimental constructs at sites predicted to interact with EcSSB. By measuring the fraction of DNA molecules with EcSSB bound as well as the volume of protein bound per DNA molecule, we determine the protein binding affinity, cooperativity, and conformation. We find that, with only one damaged nucleotide, the binding of EcSSB is unchanged relative to its binding to un-damaged DNA. In the presence of either two tandem abasic sites or a non-DNA spacer, however, the binding affinity associated with a single EcSSB tetramer occupying the full substrate in the 65-nt mode is greatly reduced. In contrast, the binding of two EcSSB tetramers, each in the 35-nt mode, is preserved. Changes in the binding and cooperative behaviors of EcSSB across these constructs can inform how genomic repair and replication processes may change as environmental damage accumulates in DNA.SIGNIFICANCE Single-stranded binding proteins (SSBs) bind transiently exposed single-stranded DNA (ssDNA) during DNA replication, recombination, and repair. SSBs both protect ssDNA from degradation and recruit additional proteins to aid in essential cellular processes. Escherichia coli SSB (EcSSB), a well-studied model system, binds ssDNA in multiple conformations, occluding variable lengths of substrate. We examine EcSSB binding to ssDNA substrates at a single-molecule level and find that modifying DNA to imitate naturally occurring DNA damage alters the preferred binding conformation of EcSSB without reducing its high binding affinity. Our results suggest that EcSSB can bind damaged ssDNA in a site-directed manner that could help facilitate specific remediation of individual bases.

Automated summary

Single-stranded DNA-binding proteins (SSBs) play a crucial role in DNA repair and replication. This study investigated the binding of SSB to individual DNA molecules and the effect of DNA damage on binding conformation. The results showed that the presence of damaged nucleotides reduced the binding affinity of SSB, but did not affect its binding capability. These findings provide insights into the changes in genomic repair and replication processes under accumulating environmental damage.