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Ordered phases of helical DNA+RecA complexes

The cell as the basic functional entity of life is a very crowded environment, where DNA has to be packed, either through nonspecific electrostatic interactions, or via specifically engineered proteins. Such dense phases, with a high volume proportion of biomacromolecules (>10%) are functional structures. E.coli RecA protein, important in homologous recombination - which takes place in the crowded environment of a chromosome - achieves its function only after polymerizing along DNA and forming nucleoprotein helical filaments. Since the strand exchange reaction mediated by RecA is supposed to occur in the dense phase, we focused on the organization of concentrated RecA and RecA/DNA complexes. RecA protein was purified from E coli and complexes were formed using short DNA fragments (146pb, 50nm), expecting to create a monodisperse system of rodlike particles. We intended to compare the supramolecular organization of double stranded DNA fragments, RecA filaments and RecA/DNA complexes. We followed the RecA/DNA complexation process using fluorescence correlation spectroscopy and we checked by electron microscopy that complexes longer than the initial 50 nm DNA fragments are formed under adequate ionic conditions (Fig. 1). These RecA/DNA complexes spontaneously form ordered phases when their concentration is increased (Fig.2) as also the RecA filaments (Fig.3). Analyses are on the way to determine the nature of these phases and whether phase transitions may be coupled to the complex formation and/or strand exchange reaction.

ordered phases; DNA; proteins; supramolecular organization; fluorescence correlation spectroscopy; electron microscopy

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