Elliott Crooke

The Initiator Function of DnaA Protein Is Negatively Regulated by the Sliding Clamp of the E. coli Chromosomal Replicase

The beta subunit of DNA polymerase III is essential for negative regulation of the initiator protein, DnaA. DnaA inactivation occurs through accelerated hydrolysis of ATP bound to DnaA; the resulting ADP-DnaA fails to initiate replication. The ability of beta subunit to promote DnaA inactivation depends on its assembly as a sliding clamp on DNA and must be accompanied by a partially purified factor, IdaB protein. DnaA inactivation in the presence of IdaB and DNA polymerase III is further stimulated by DNA synthesis, indicating close linkage between initiator inactivation and replication. In vivo, DnaA predominantly takes on the ADP form in a beta subunit-dependent manner. Thus, the initiator is negatively regulated by action of the replicase, a mechanism that may be key to effective control of the replication cycle.

Escherichia coli prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA.

Initiator DnaA and DNA bending proteins, Fis and IHF, comprise prereplication complexes (pre‐RC) that unwind the Escherichia coli chromosomes origin of replication, oriC. Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli. Based on dimethylsulphate (DMS) footprinting of purified proteins, we observed a dynamic interplay among Fis, IHF and DnaA on supercoiled oriC templates. Low levels of Fis inhibited oriC unwinding by blocking both IHF and DnaA binding to low affinity sites. As the concentration of DnaA was increased, Fis repression was relieved and IHF rapidly redistributed DnaA to all unfilled binding sites on oriC. This behaviour in vitro is analogous to observed assembly of pre‐RC in synchronized E. coli. We propose that as new DnaA is synthesized in E. coli, opposing activities of Fis and IHF ensure an abrupt transition from a repressed complex with unfilled weak affinity DnaA binding sites to a completely loaded unwound complex, increasing both the precision of DNA replication timing and initiation synchrony.

Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication

Initiation of DNA replication from the Escherichia coli chromosomal origin is highly regulated, assuring that replication occurs precisely once per cell cycle. Three mechanisms for regulation of replication initiation have been proposed: titration of free DnaA initiator protein by the datA locus, sequestration of newly replicated origins by SeqA protein and regulatory inactivation of DnaA (RIDA), in which active ATP‐DnaA is converted to the inactive ADP‐bound form. DNA microarray analyses showed that the level of initiation in rapidly growing cells that lack datA was indistinguishable from that in wild‐type cells, and that the absence of SeqA protein caused only a modest increase in initiation, in agreement with flow‐cytometry data. In contrast, cells lacking Hda overinitiated replication twofold, implicating RIDA as the predominant mechanism preventing extra initiation events in a cell cycle.

CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli

CspD is a stationary phase‐induced, stress response protein in the CspA family of Escherichia coli. Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication. CspD consists mainly of β‐strands, and the purified protein exists exclusively as a dimer and binds to single‐stranded (ss)DNA and RNA in a dose‐dependent manner without apparent sequence specificity. CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro. Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB‐coated DNA. We propose that CspD dimers, with two independent β‐sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient‐depleted cells.

Mutations in DnaA protein suppress the growth arrest of acidic phospholipid-deficient Escherichia coli cells.

Cell growth arrests when the concentrations of anionic phospholipids drop below a critical level in Escherichia coli, with the insufficient amounts of acidic phospholipids adversely affecting the DnaA‐dependent initiation of DNA replication at the chromosomal origin (oriC). Mutations have been introduced into the carboxyl region of DnaA, including the portion identified as essential for productive in vitro DnaA‐acidic phospholipid interactions. Expression of DnaA proteins possessing certain small deletions or substituted amino acids restored growth to cells deficient in acidic phospholipids, whereas expression of wild‐type DnaA did not. The mutations include substitutions and deletions in the phospholipid‐interacting domain as well as some small deletions in the DNA‐binding domain of DnaA. Marker frequency analysis indicated that initiation of replication occurs at or near oriC in acidic phospholipid‐ deficient cells rescued by the expression of DnaA having a point mutation in the membrane‐binding domain, DnaA(L366K). Flow cytometry revealed that expression in wild‐type cells of plasmid‐borne DnaA(L366K) and DnaA(Δ363–367) reduced the frequency with which replication was initiated and disturbed the synchrony of initiations.

Characterization of Escherichia coli DnaAcos protein in replication systems reconstituted with highly purified proteins

Excessive initiation of chromosomal replication occurs in the dnaAcos mutant at 30°C. Whereas purified wild‐type DnaA protein binds ATP and ADP tightly, DnaAcos protein is defective for such nucleotide binding. As initiation is a multistep reaction and DnaA protein functions at each step, activities of DnaAcos protein need to be examined precisely. DnaAcos protein specifically bound a DNA fragment containing the chromosomal replication origin with an affinity similar to that seen with the wild‐type protein. In a system reconstituted with purified proteins at 30°C, the mutant protein initiated replication of single‐stranded DNA that contains a DnaA‐binding hairpin structure. Thus, DnaAcos protein basically sustains affinity to a DnaA‐binding sequence and functions in the loading of DnaB helicase onto single‐stranded DNA. Thermal stabilities of wild‐type DnaA and DnaAcos activities were comparable. Unlike wild‐type DnaA protein, DnaAcos protein was inactive for minichromosomal replication in systems reconstituted with purified proteins in which the ATP‐bound form of DnaA protein is required for initiation. Taken together, the data indicate that the prominent defect in DnaAcos protein appears to be the inability to bind nucleotide.

Restoration of Growth to Acidic Phospholipid-deficient Cells by DnaA(L366K) Is Independent of Its Capacity for Nucleotide Binding and Exchange and Requires DnaA

In the absence of adequate levels of cellular acidic phospholipids, Escherichia coli remain viable but are arrested for growth. Expression of a DnaA protein that contains a single amino acid substitution in the membrane-binding domain, DnaA(L366K), in concert with expression of wild-type DnaA protein, restores growth. DnaA protein has high affinity for ATP and ADP, and in vitro lipid bilayers that are fluid and contain acidic phospholipids reactivate inert ADP-DnaA by promoting an exchange of ATP for ADP. Here, nucleotide and lipid interactions and replication activity of purified DnaA(L366K) were examined to gain insight into the mechanism of how it restores growth to cells lacking acidic phospholipids. DnaA(L366K) behaved like wild-type DnaA with respect to nucleotide binding affinities and hydrolysis properties, specificity of acidic phospholipids for nucleotide release, and origin binding. Yet, DnaA(L366K) was feeble at initiating replication from oriC unless augmented with a limiting quantity of wild-type DnaA, reflecting the in vivo requirement that both wild-type and a mutant form of DnaA must be expressed and act together to restore growth to acidic phospholipid deficient cells.

Remodeling of Nucleoprotein Complexes Is Independent of the Nucleotide State of a Mutant AAA+ Protein

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.

Nucleotide-Induced Conformational Changes in Escherichia coli DnaA Protein Are Required for Bacterial ORC to Pre-RC Conversion at the Chromosomal Origin

DnaA oligomerizes when bound to origins of chromosomal replication. Structural analysis of a truncated form of DnaA from Aquifex aeolicus has provided insight into crucial conformational differences within the AAA+ domain that are specific to the ATP- versus ADP- bound form of DnaA. In this study molecular docking of ATP and ADP onto Escherichia coli DnaA, modeled on the crystal structure of Aquifex aeolicus DnaA, reveals changes in the orientation of amino acid residues within or near the vicinity of the nucleotide-binding pocket. Upon limited proteolysis with trypsin or chymotrypsin ADP-DnaA, but not ATP-DnaA generated relatively stable proteolytic fragments of various sizes. Examined sites of limited protease susceptibility that differ between ATP-DnaA and ADP-DnaA largely reside in the amino terminal half of DnaA. The concentration of adenine nucleotide needed to induce conformational changes, as detected by these protease susceptibilities of DnaA, coincides with the conversion of an inactive bacterial origin recognition complex (bORC) to a replication efficient pre-replication complex (pre-RC) at the E. coli chromosomal origin of replication (oriC).

Systems Medicine: A New Model for Health Care

In 2003, Dr. Elias Zerhouni created the National Institutes of Health (NIH) roadmap and articulated an agenda to aggressively pursue a more integrated approach to use research discoveries to impact human health. Working groups focused on three major themes: New Pathways to Discovery, Research Teams of the Future, and Reengineering the Clinical Research Enterprise. The findings illustrated the need to develop science to decipher biological networks, and the need for broad-scale application of bioinformatics and computational methods to biological systems [1]. In March 2007, the Department of Health and Human Services launched the Personalized Health Care Initiative (PHCI) with the aim to accelerate the development of personalized treatment strategies. The program focuses on high-throughput technologies and developing an infrastructure to promote electronic medical records [2].