Direct and real-time visualization of the disassembly of a single RecA-DNA-ATPgammaS complex using AFM imaging in fluid.

Abstract

RecA disassembly from circular double-stranded DNA (dsDNA) was studied by atomic force microscopy (AFM) imaging in fluid on a single molecule scale. The RecA/DNA complex was formed in the presence of ATPgammaS, and the disassembly was then initiated by buffer exchange to rinse off ATPgammaS. Performing AFM imaging in fluid allowed direct and real-time visualization of the disassembly of RecA from dsDNA in solution. It was found that RecA disassembly commenced from multiple sites both in deionized water and in buffer; the areas where RecA dissociated showed the appearance of "gaps" in the filamentous structure. RecA further disassembled either through the already existing "gaps" or by generation of new gaps. The disassembly was slower in buffer than in deionized water, suggesting that ions also contribute to the stabilization of the complex. RecA hexamers and monomers were observed in deionized water and in buffer, respectively, during the disassembly process.