Kinetic Assay and Visualization of Interaction of Single-Stranded DNA-Binding Protein with its Substrate
SSB蛋白与ssDNA相互作用的动力学和可视化研究

Study of interaction of E.coli single-stranded DNA-binding protein (SSB) with its substrate, single-stranded DNA (ssDNA), is very important for understanding its essential roles in replication, recombination and repair of DNA. In this report, interaction of SSB with ssDNA was monitored by surface plasmon resonance (SPR) and directly observed using atomic force microscopy (AFM), which presented an attempt to investigate the binding mode of SSB by a single-molecule visualization methodology. The resulting SSB protein was a correctly folded tetramer with an apparent binding to 43-met ssDNA with the equilibrium dissociation constant (KD) of 9.67×10-7 M and 4.79×10-7 M respectively whether MgCl2 was present or not as determined by SPR. In the AFM images, individual ssDNA, SSB protein and SSB-ssDNA complex were visualized. The used ssDNA was long enough for cooperative SSB binding and the authors observed that SSB protein distributed on ssDNA non-specifically.