Single molecule analysis ofThermus thermophilusSSB protein dynamics on single-stranded DNA

Abstract

Single-stranded (ss) DNA binding (SSB) proteins play central roles in DNA replication, recombination and repair in all organisms. We previously showed that Escherichia coli (Eco) SSB, a homotetrameric bacterial SSB, undergoes not only rapid ssDNA-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssDNA. Whereas the majority of bacterial SSB family members function as homotetramers, dimeric SSB proteins were recently discovered in a distinct bacterial lineage of extremophiles, the Thermus–Deinococcus group. Here we show, using single-molecule fluorescence resonance energy transfer (FRET), that homodimeric bacterial SSB from Thermus thermophilus (Tth) is able to diffuse spontaneously along ssDNA over a wide range of salt concentrations (20–500 mM NaCl), and that TthSSB diffusion can help transiently melt the DNA hairpin structures. Furthermore, we show that two TthSSB molecules undergo transitions among different DNA-binding modes while remaining bound to ssDNA. Our results extend our previous observations on homotetrameric SSBs to homodimeric SSBs, indicating that the dynamic features may be shared among different types of SSB proteins. These dynamic features of SSBs may facilitate SSB redistribution and removal on/from ssDNA, and help recruit other SSB-interacting proteins onto ssDNA for subsequent DNA processing in DNA replication, recombination and repair.

Real-time submillisecond single-molecule FRET dynamics of freely diffusing molecules with liposome tethering

“…These results show that our method would be very useful for investigating fast dynamics of homodimeric SSBs. In addition, our results together with previous work by Ha and colleagues 46 58 indicate that the diffusional migration or redistribution of SSB on ssDNA is a general phenomenon. The diffusional migration of dr SSB is much faster than that of tetrameric E. coli SSB.…”

“…Recently, Ha and colleagues 46 58 have studied the dynamics of Thermus thermophilus ( tt ) SSB on ssDNA. tt SSB is a homodimeric SSB similar to dr SSB.…”

The impact of base stacking on the conformations and electrostatics of single-stranded DNA

“…It seems difficult to motivate an orders-of-magnitude difference in binding affinity based solely from the presence of additional base stacks, which are likely transiently breaking and reforming. Furthermore, many SSB's such as replication protein A (RPA) and Escherichia coli (Eco) SSB can melt DNA secondary structure while diffusing on long strands of poly dT ( 56 , 57 ). These results appear to be inconsistent, suggesting on the one hand that base stacking can hamper binding, while on the other, these proteins show behavior that is unaffected by DNA structure.…”

Molecular determinants of the interactions between proteins and ssDNA

“…Similarly, RPA can bind very tightly to an ssDNA and dissociate rapidly when other SSBs are present in solution (20). Unlike studies (10,15,16,19) that were performed on an infinitely dilute solution, the findings of the study in ref. 20 apply to a wide range of conditions.…”

“…Recent studies using single-molecule fluorescence resonance energy transfer have endeavored to shed light on the dynamic activity of different types of SSBs, which were earlier thought to form inert complexes with ssDNAs. These studies have shown that the Escherichia coli SSB functions as a sliding platform that migrates on ssDNA (10,15), whereas a homodimeric bacterial SSB from Thermus thermophilus is able to diffuse spontaneously along ssDNA (16). It was reported that the diffusion coefficient of the E. coli SSB along ssDNA is similar to that of transcription factors along dsDNA (17).…”