Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering
The importance of filament formation of RecA for the DNA strand exchange reaction, both in vivo and in vitro, is established. RecA forms a very long and relatively stiff filament by binding around DNA with high cooperativity. The monomer units are assembled in the filament in a head-to-tail arrangement in a helical manner, similar to the organization of RecA molecules found in the crystal of pure RecA or including ADP. This filament of RecA, containing a DNA molecule in its interior, can bind another DNA molecule and yet a third one in the presence of cofactor (ATP or its analogs). Each filament may have three DNA binding sites, each able to bind one DNA strand of either ss or ds DNA. According to linear dichroism and fluorescence spectroscopies, the DNA molecules in the RecA filament are well organized with a well defined but modified structure. This organization and modification of DNA by RecA probably has the purpose of facilitating Watson-Crick base-pair recognition and strand exchange reaction. RecA is thus actively involved in the reaction. The phosphoribose backbone of DNA follows the RecA helix and the DNA is stretched 50% and unwound. The nucleobases are destacked but still firmly oriented almost perpendicular to the axis for the first DNA and are immobile even in the case of ssDNA. Even in the second DNA the motion of DNA bases is very restricted although their orientation appears to be less perpendicular. All DNA strands in the complex show some sequence dependent interaction with each other. It could be a non-conventional base-base pairing, that could provide a mechanism of search for homologous DNA. In order to understand how RecA organizes DNA, a 3-D model building of the RecA-DNA complex is in progress undertaken based on the crystal structure of RecA and results obtained using chemical interference and protein engineering techniques. A characterization of structure of the second DNA and the mode of DNA-DNA interaction may further clarify the reaction mechanism of strand exchange.