engleski

Packing of monodisperse DNA-recA protein complexes

We study E.coli RecA protein, from preparation and purification of protein monomers to structural studies of protein polymers formed on DNA – nucleoprotein filaments. This protein is a multirole one, where DNA strand exchange by forming nucleoprotein filament during homologous recombination and cleavage of SOS response repressors are the most prominent two. The first role, where we are interested into structural details (and structure makes function, very much so in this case) also came recently into focus with the work of Zahradka et al.[1], where reassembly of shattered chromosomes in D. radiodurans is completed by RecA-dependent crossovers. RecA is a relatively small protein, MW = 37, 842, with 352 amino acid residues. Only by polymerizing within nucleoprotein filaments it achieves its function. The RecA polymers have been crystallized and structure determined to atomic resolution by XRD [2], however the structure of RecA-DNA complex has only been studied by electron microscopy or NMR, and the exact path of DNA within the nucleoprotein filament is not known [3]. We plan to produce liquid crystals of RecA nucleoprotein filaments using very short, monodisperse, 146 bp long DNA [4]. Such 50~75 nm long filaments will be shorter than their respective persistence length – i.e. they will behave as straight rods. Presumably, an ordered nucleoprotein filament phase, studied by optical and electronic microscopies and XRD will allow further insight into the function of RecA, especially if a correlation will be found between the length of the filaments and their activity. [1] Zahradka K., Slade D., Bailone A., Sommer S., Averbeck D., Petranovic M., Lindner A.B. & Radman M., Nature 443: 569-573 (2006). [2] Story R. M., Weber I. T. & Steitz T. A., Nature 355: 318– 325 (1992). [3] Yu X., Jacobs S.A., West S.C., Ogawa T. & Egelman E.H., PNAS 98: 8419-8424 (2001). [4] Sikorav J.-L., Pelta J. & Livolant F., Biophys. J. 67:1387– 1392 (1994).

RecA protein; liquid crystals; persistence length; protein-DNA interaction

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