Elastic motion of RecA filaments and its role in recombination.

RecA filaments form two types of structures having different helical pitches according to the nucleotide condition. We have investigated the effect of nucleotide cofactor exchange upon the RecA-ssDNA filaments by observing a fluorescently-labelled single molecule in water solution. The result indicated that the RecA-ssDNA filamentous complex is an elastic helical molecule whose length is controlled by binding and release of nucleotide cofactors. We propose that this elastic motion couples to the DNA rotation within the filament by synchronizing the helical phases, and promotes exchange of homologous strands of two DNAs.

Liquid crystal formation of RecA-DNA filamentous complexes.

Spontaneous optical birefringence of RecA-bound linear and closed circular single-stranded DNA filaments, as well as RecA self-assembled polymer, was observed in aqueous buffer solutions, which demonstrates the formation of lyotropic liquid crystalline phases.

Elastic behavior of RecA-DNA helical filaments.

Escherichia coli RecA protein forms a right-handed helical filament with DNA molecules and has an ATP-dependent activity that exchanges homologous strands between single-stranded DNA (ssDNA) and duplex DNA. We show that the RecA-ssDNA filamentous complex is an elastic helical molecule whose length is controlled by the binding and release of nucleotide cofactors. RecA-ssDNA filaments were fluorescently labelled and attached to a glass surface inside a flow chamber. When the chamber solution was replaced by a buffer solution without nucleotide cofactors, the RecA-ssDNA filament rapidly contracted approximately 0.68-fold with partial filament dissociation. The contracted filament elongated up to 1.25-fold when a buffer solution containing ATPgammaS was injected, and elongated up to 1.17-fold when a buffer solution containing ATP or dATP was injected. This contraction-elongation behavior was able to be repeated by the successive injection of dATP and non-nucleotide buffers. We propose that this elastic motion couples to the elastic motion and/or the twisting rotation of DNA strands within the filament by adjusting their helical phases.

Visualization of RecA filaments and DNA by fluorescence microscopy.

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.

Conductive metal nanowires templated by the nucleoprotein filaments, complex of DNA and RecA protein.

Development of preprogrammable conductive nanowires is a requisite for the future fabrication of nanoscale electronics based on molecular assembly. Here, we report the synthesis of conductive metal nanowires from nucleoprotein filaments, complexes of single- or double-stranded DNA and RecA protein. A genetically engineered RecA derivative possessing a reactive and surface accessible cysteine residue was reacted with functionalized gold particles, resulting in nucleoprotein filaments with gold particles attached. The template-based gold particles were enlarged by chemical deposition to form uniformly metallized nanowires. The programming information can be encoded in DNA sequences so that an intricate electrical circuit can be constructed through self-assembly of each component. As the RecA filament has higher degree of stiffness than double-stranded DNA, it provides a robust scaffold that allows us to fabricate more reliable and well-organized electrical circuitry at the nanoscale. Furthermore, the function of homologous pairing provides sequence-specific junction formation as well as sequence-specific patterning metallization.