title: Remodeling of nucleoprotein complexes is independent of the nucleotide state of a mutant AAA+ protein.
abstract: DnaA protein, a member of the AAA+ (ATPase associated with various cellular activities) family, initiates DNA synthesis at the chromosomal origin of replication (oriC) and regulates the transcription of several genes, including its own. The assembly of DnaA complexes at chromosomal recognition sequences is affected by the tight binding of ATP or ADP by DnaA. DnaA with a point mutation in its membrane-binding amphipathic helix, DnaA(L366K), previously described for its ability to support growth in cells with altered phospholipid content, has biochemical characteristics similar to those of the wild-type protein. Yet DnaA(L366K) fails to initiate in vitro or in vivo replication from oriC. We found here, through in vitro dimethyl sulfate footprinting and gel mobility shift assays, that DnaA(L366K) in either nucleotide state was unable to assemble into productive prereplication complexes. In contrast, at the dnaA promoter, both the ATP and the ADP form of DnaA(L366K) generated active nucleoprotein complexes that efficiently repressed transcription in a manner similar to wild-type ATP-DnaA. Thus, it appears that unlike wild-type DnaA protein DnaA(L366K) can adopt architectures that are independent of its bound nucleotide, and instead the locus determines the functionality of the higher order DnaA(L366K)-DNA complexes.
authors.affiliation: Department of Biochemistry and Molecular and Cellular Biology, Georgetown University Medical Center, Washington, DC 20007, USA.
authors.forename: Rahul
authors.initials: R
authors.lastname: Saxena
authors.forename: Tania
authors.initials: T
authors.lastname: Rozgaja
authors.forename: Julia
authors.initials: J
authors.lastname: Grimwade
authors.forename: Elliott
authors.initials: E
authors.lastname: Crooke
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology; D004269:DNA, Bacterial / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D014158:Transcription, Genetic / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D009097:Multienzyme Complexes; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D000255:Adenosine Triphosphate