DNA linking number change induced by sequence-specific DNA-binding proteins.

Sequence-specific DNA-binding proteins play a key role in many fundamental biological processes, such as transcription, DNA replication and recombination. Very often, these DNA-binding proteins introduce structural changes to the target DNA-binding sites including DNA bending, twisting or untwisting and wrapping, which in many cases induce a linking number change (Delta Lk) to the DNA-binding site. Due to the lack of a feasible approach, Delta Lk induced by sequence-specific DNA-binding proteins has not been fully explored. In this paper we successfully constructed a series of DNA plasmids that carry many tandem copies of a DNA-binding site for one sequence-specific DNA-binding protein, such as lambda O, LacI, GalR, CRP and AraC. In this case, the protein-induced Delta Lk was greatly amplified and can be measured experimentally. Indeed, not only were we able to simultaneously determine the protein-induced Delta Lk and the DNA-binding constant for lambda O and GalR, but also we demonstrated that the protein-induced Delta Lk is an intrinsic property for these sequence-specific DNA-binding proteins. Our results also showed that protein-mediated DNA looping by AraC and LacI can induce a Delta Lk to the plasmid DNA templates. Furthermore, we demonstrated that the protein-induced Delta Lk does not correlate with the protein-induced DNA bending by the DNA-binding proteins.