PARTNER SWITCHING MECHANISMS IN INACTIVATION AND REJUVENATION OF ESCHERICHIA-COLI DNA GYRASE BY F-PLASMID PROTEINS LETD (CCDB) AND LETA (CCDA)

Escherichia coil DNA gyrase, as well as a free form of its A subunit ( GyrA), exists in an inactivated form in cells that overproduce the F p lasmid protein LetD (CcdB). We found that the inactivated DNA gyrase a nd GyrA protein can be rejuvenated in vitro by another F plasmid prote in, LetA (CcdA). Using this rejuvenation as an assay, we purified the inactivated GyrA protein to near homogeneity and found it to be comple xed with the LetD protein. The complex has a molecular mass of 230 kDa and was suggested to be a complex of two molecules each of GyrA and L etD proteins. The GyrA-LetD complex, in the presence of purified GyrB protein, does not cause DNA cleavage. Therefore, the LetD protein in t he GyrA-LetD complex inhibits the gyrase action by a mechanism differe nt from one that involves trapping a covalently linked gyrase-DNA comp lex. In as much as a free form of the LetD protein has been shown to i nduce DNA cleavage by gyrase, the LetD protein seems to have two disti nct modes of action on DNA gyrase. Rejuvenation of the inactivated Gyr A protein by the LetA protein was achieved in vitro, and mechanisms go verning this process were examined using the purified proteins. The re juvenated GyrA protein sediments through sucrose gradients as a single protein species of 190 kDa and is indistinguishable from a free form of GyrA protein. In the same sedimentation experiment, the LetD protei n was seen to be complexed with the added LetA protein. Thus, the LetA protein apparently rejuvenates the GyrA protein by removing the bound LetD protein from the inactivated form, followed by formation of a Le tA-LetD complex. (C) 1996 Academic Press Limited