Atomic force microscopic study of low temperature induced disassembly of RecA-dsDNA filaments

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.