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 wildtype 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.

Differentiation of the DnaA-oriC Subcomplex for DNA Unwinding in a Replication Initiation Complex

“…Walker A/B, sensor 1/2, and an Arg finger) and plays a crucial role in ATP/ADP binding/hydrolysis, inter-DnaA interaction, and DUE unwinding (4,45,46). Domain IV has a DNA-binding helix-turn-helix motif (46,47), in addition to an amphiphilic helix, which is connected to domain III and might be important for inter-DnaA interaction (48).…”

The initiator protein DnaA, chromosomal origin oriC and their interaction to generate origin recognition complex and pre-replication complex like nucleoprotein structures in Escherichia coli

“…However, substitutions at the first two positions within high affinity R boxes weaken their interaction with either ADP-DnaA or ATPDnaA and thus fail to promote origin unwinding 35 . In contrast to high affinity DnaA recognition sequences, low affinity sites prefer interaction with only ATP-DnaA 12,32 . Interestingly, this specificity can be eliminated by mutating the guanine at the third position of I2 and I3 12 .…”

“…Filter retentions 9 , gel mobility shift 37 , Dnase I footprint 32,38 and di-methyl sulphate footprint 12,32 assays have revealed different functional aspects of the interaction between DnaA protein and chromosomal origin. It is to be noted that the stoichometery of DnaA bound to oriC varies depending on the method of detection 8,9,37,39 .…”

“…Beside the specific arrangement of DnaA binding elements present within oriC, a mutation, DnaA(L366K) 46 , present in the membrane binding domain of DnaA protein (Figure 1) also prevents ORC to pre-RC conversion 32 . In-fact in vitro, an ADP-DnaA ORC can not support the loading of ATP-DnaA(L366K) to low affinity sites 32 .…”

“…In-fact in vitro, an ADP-DnaA ORC can not support the loading of ATP-DnaA(L366K) to low affinity sites 32 . On the other hand, an ADPDnaA(L366K) ORC can successfully be converted to a pre-RC in the presence of ATP-DnaA protein 32 . These properties of DnaA(L366K) protein has been attributed to its altered interaction with low affinity recognition sequences.…”