Partner switching mechanisms in inactivation and rejuvenation of Escherichia coli DNA gyrase by F plasmid proteins LetD (CcdB) and LetA (CcdA).
Escherichia coli DNA gyrase, as well as a free form of its A subunit (GyrA), exists in an inactivated form in cells that overproduce the F plasmid protein LetD (CcdB). We found that the inactivated DNA gyrase and GyrA protein can be rejuvenated in vitro by another F plasmid protein, LetA (CcdA). Using this rejuvenation as an assay, we purified the inactivated GyrA protein to near homogeneity and found it to be complexed 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 LetD proteins. The GyrA-LetD complex, in the presence of purified GyrB protein, does not cause DNA cleavage. Therefore, the LetD protein in the GyrA-LetD complex inhibits the gyrase action by a mechanism different from one that involves trapping a covalently linked gyrase-DNA complex. In as much as a free form of the LetD protein has been shown to induce DNA cleavage by gyrase, the LetD protein seems to have two distinct modes of action on DNA gyrase. Rejuvenation of the inactivated GyrA protein by the LetA protein was achieved in vitro, and mechanisms governing this process were examined using the purified proteins. The rejuvenated 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 protein 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 LetA-LetD complex.
Maki
S
S
Faculty of Pharmaceutical Sciences, Kyushu Univeristy, Fukuoka, Japan.
Takiguchi
S
S
Horiuchi
T
T
Sekimizu
K
K
Miki
T
T
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D005144:F Factor; D008970:Molecular Weight; D011485:Protein Binding
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068355:CcdA protein, Bacteria; C068356:CcdB protein, Plasmid F; D004247:DNA; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II