Atomic force microscopic study of low temperature induced disassembly of RecA-dsDNA filaments.
ABSTRACT The assembly and disassembly of RecA-DNA nucleoprotein filaments on double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) are important steps for homologous recombination and DNA repair. The assembly and disassembly of the nucleoprotein filaments are sensitive to the reaction conditions. In this work, we investigated different morphologies of the formed nucleoprotein filaments at low temperature under different solution conditions by atomic force microscopy (AFM). We found that low temperature and long keeping time could induce the incomplete disassembly of the formed nucleoprotein filaments. In addition, when the formed filaments were kept at -20 degrees C for 20 h with 1,4-dithiothreitol (DTT), the integrated filaments disassembled. It was similar to the case under the same condition without anything added. However, when glycerol was used as a substitute for DTT, there was no obvious disassembly at the same condition. Oppositely, when the formed filaments were kept at 4 degrees C for 20 h, the disassembly with additional DTT was not as obvious as the case at -20 degrees C for 20 h, whereas the case with additional glycerol disassembled. The experiments indicated the effect of cold denaturation on the interaction of DNA and RecA. Meanwhile, the study of these phenomena can supply guidelines for the property and stability of RecA as well as the relevant roles of influencing factors to RecA and DNA in further theoretical studies.
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ABSTRACT: By exploring a recent model in which DNA bending elasticity, described by the wormlike chain model, is coupled to basepair denaturation, we demonstrate that small denaturation bubbles lead to anomalies in the flexibility of DNA at the nanometric scale, when confined in two dimensions (2D), as reported in atomic-force microscopy experiments. Our model yields very good fits to experimental data and quantitative predictions that can be tested experimentally. Although such anomalies exist when DNA fluctuates freely in three dimensions (3D), they are too weak to be detected. Interactions between bases in the helical double-stranded DNA are modified by electrostatic adsorption on a 2D substrate, which facilitates local denaturation. This work reconciles the apparent discrepancy between observed 2D and 3D DNA elastic properties and points out that conclusions about the 3D properties of DNA (and its companion proteins and enzymes) do not directly follow from 2D experiments by atomic-force microscopy.Biophysical Journal 07/2009; 96(11):4464-9. · 3.67 Impact Factor
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ABSTRACT: Disassembly of DNA-ligands, including DNA-methylene blue (MB) complex and DNA-Co(phen)(3) (3+) complex on mica surface, were investigated by atomic force microscopy (AFM). The disassembly of these complexes occurred after they were immersed in ultra-pure water. AFM results showed that the disassembly depended strongly on bridge ions that were used to immobilize the complex onto mica surface, DNA species and ligands. When Mg(2+) was used as the bridge ion, the DNA-MB complex was completely disassembled because of the weak interactions between Mg(2+) and DNA's bases or mica surface. Although if Co(2+) was used as the bridge ion, the disassembly of the DNA-MB complex mainly depended on the species and shape of DNA. For plasmid DNA pBR 322, plasmid DNA pUC 18 and the linear DNA pBR 322/PstI, the degree of disassembly was gradually increased. Whereas if Co(phen)(3) (3+) was chosen as the ligand, the disassembly of the DNA-Co(phen)(3) (3+) complex was almost blocked because Co(phen)(3) (3+) could hardly diffuse into the ultra-pure water. This obtained information may be useful for practical application of the AFM imaging of biological molecules, especially in liquid.Journal of Microscopy 06/2009; 234(2):130-6. · 1.63 Impact Factor