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- W2010718744 abstract "Protein-DNA interactions play a key role in many fundamental biological processes, such as DNA replication, transcription, and recombination. Very often, the DNA-binding proteins induce structural changes of the target DNA-binding sequences. These DNA structural changes include DNA bending, twisting, and wrapping. Although protein-induced DNA-bending has been widely studied, other protein-induced DNA structural changes have not been fully explored due to the lack of a feasible experimental approach. In this study, we developed a new experimental strategy to probe the protein-induced DNA-unwinding. Our first step is to make a circular plasmid DNA template containing multiple tandem copy of a DNA-binding site of a sequence-specific DNA-binding protein, such as λ O protein and LacI. This plasmid also contains one nicking-enzyme recognition site, such as Nb.Bsm I. Next, we nicked this plasmid using the nicking enzyme, Nb.Bsm I, and then titrated the nicked plasmid by increasing the concentration of the DNA-binding protein. The DNA template was ligated by T4 DNA ligase. The linking number (Lk) of the ligated DNA products was determined by one or two-dimensional agarose gel electrophoresis in the presence of chloroquine. The linking number changes (ΔLK) was calculated using equation ΔLK=Lk-Lk°, where Lk and Lk° are the Boltzmann centers of the topoisomers band in the presence of the DNA-binding protein and in its absence, respectively. If we define the apparent protein-induced DNA unwinding angle (αobs) equals to 360×ΔLK, i.e. αobs =360× ΔLK, then the protein-induced DNA-unwinding angle (α) and the DNA-binding constant (K) can be simultaneously determined. Using this method, we have determined the DNA-unwinding angle and the DNA-binding constant for the following sequence-specific DNA-binding protein: λO, LacI, GalR, AraC, and CRP. Our results indicate that protein-induced DNA-unwinding is a unique feature for these sequence-specific DNA-binding proteins." @default.
- W2010718744 created "2016-06-24" @default.
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- W2010718744 date "2009-02-01" @default.
- W2010718744 modified "2023-09-29" @default.
- W2010718744 title "Protein-Induced DNA Unwinding is An Intrinsic Feature of Certain Sequence-Specific DNA-Binding Proteins" @default.
- W2010718744 doi "https://doi.org/10.1016/j.bpj.2008.12.2115" @default.
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