The linker domain of the initiator DnaA contributes to its ATP binding and membrane association in chromosomal replication.

DnaA, the initiator of chromosomal replication, has in its adenosine triphosphatase (ATPase) domain residues required for adenosine 5'-triphosphate (ATP) binding and membrane attachment. Here, we show that D118Q substitution in the DnaA linker domain, a domain known to be without major regulatory functions, influences ATP binding of DnaA and replication initiation in vivo. Although initiation defective by itself, overexpression of DnaA(D118Q) caused overinitiation of replication in ts cells and prevented cell growth.

Membrane Stress Caused by Unprocessed Outer Membrane Lipoprotein Intermediate Pro-Lpp Affects DnaA and Fis-Dependent Growth.

In , repression of phosphatidylglycerol synthase A gene () lowers the levels of membrane acidic phospholipids, particularly phosphatidylglycerol (PG), causing growth-arrested phenotype. The interrupted synthesis of PG is known to be associated with concomitant reduction of chromosomal content and cell mass, in addition to accumulation of unprocessed outer membrane lipoprotein intermediate, pro-Lpp, at the inner membrane. However, whether a linkage exists between the two altered-membrane outcomes remains unknown.

Inorganic polyphosphate in platelet rich plasma accelerates re-epithelialization and .

Wound healing requires well-coordinated events including hemostasis, inflammation, proliferation, and remodeling. Delays in any of these stages leads to chronic wounds, infections, and hypertrophic scarring. Burn wounds are particularly problematic, and may require intervention to ensure timely progression to reduce morbidity and mortality.

A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin.

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution.

A Novel Inositol Pyrophosphate Phosphatase in Saccharomyces cerevisiae: Siw14 PROTEIN SELECTIVELY CLEAVES THE β-PHOSPHATE FROM 5-DIPHOSPHOINOSITOL PENTAKISPHOSPHATE (5PP-IP5).

Inositol pyrophosphates are high energy signaling molecules involved in cellular processes, such as energetic metabolism, telomere maintenance, stress responses, and vesicle trafficking, and can mediate protein phosphorylation. Although the inositol kinases underlying inositol pyrophosphate biosynthesis are well characterized, the phosphatases that selectively regulate their cellular pools are not fully described. The diphosphoinositol phosphate phosphohydrolase enzymes of the Nudix protein family have been demonstrated to dephosphorylate inositol pyrophosphates; however, theSaccharomyces cerevisiaehomolog Ddp1 prefers inorganic polyphosphate over inositol pyrophosphates.

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.

Crosstalk between DnaA protein, the initiator of Escherichia coli chromosomal replication, and acidic phospholipids present in bacterial membranes.

Anionic (i.e., acidic) phospholipids such as phosphotidylglycerol (PG) and cardiolipin (CL), participate in several cellular functions.

Depletion of acidic phospholipids influences chromosomal replication in Escherichia coli.

In Escherichia coli, coordinated activation and deactivation of DnaA allows for proper timing of the initiation of chromosomal synthesis at the origin of replication (oriC) and assures initiation occurs once per cell cycle. In vitro, acidic phospholipids reactivate DnaA, and in vivo depletion of acidic phospholipids, results in growth arrest. Growth can be restored by the expression of a mutant form of DnaA, DnaA(L366K), or by oriC-independent DNA synthesis, suggesting acidic phospholipids are required for DnaA- and oriC-dependent replication.

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.

Polymerase chaperoning and multiple ATPase sites enable the E. coli DNA polymerase III holoenzyme to rapidly form initiation complexes.

Cellular replicases include three subassemblies: a DNA polymerase, a sliding clamp processivity factor, and a clamp loader complex. The Escherichia coli clamp loader is the DnaX complex (DnaX(3)δδ'χψ), where DnaX occurs either as τ or as the shorter γ that arises by translational frameshifting. Complexes composed of either form of DnaX are fully active clamp loaders, but τ confers important replicase functions including chaperoning the polymerase to the newly loaded clamp to form an initiation complex for processive replication.

Laminin-1 induces E-cadherin expression in 3-dimensional cultured breast cancer cells by inhibiting DNA methyltransferase 1 and reversing promoter methylation status.

This study determines the role of laminin-1 in promoting metastatic colonization during breast cancer. For this purpose, human mammary epithelial cell lines representing normal (MCF-10A), adenocarcinoma (MCF-7), and malignant carcinoma (MDA-MB-231) were propagated in 3-dimensional cultures composed of laminin-1, collagen I, or mixtures of the two, and analyzed by Western blot, immunocytochemistry, semiquantitative reverse transcription polymerase chain reaction, and methylation-specific PCR. Here we demonstrate that laminin-1 decreases methylation of the E-cadherin promoter, resulting in increased mRNA and protein expression for malignant mammary epithelial cells.

Escherichia coli DnaA forms helical structures along the longitudinal cell axis distinct from MreB filaments.

DnaA initiates chromosomal replication in Escherichia coli at a well-regulated time in the cell cycle. To determine how the spatial distribution of DnaA is related to the location of chromosomal replication and other cell cycle events, the localization of DnaA in living cells was visualized by confocal fluorescence microscopy. The gfp gene was randomly inserted into a dnaA-bearing plasmid via in vitro transposition to create a library that included internally GFP-tagged DnaA proteins.

Organization of sister origins and replisomes during multifork DNA replication in Escherichia coli.

The replication period of Escherichia coli cells grown in rich medium lasts longer than one generation. Initiation thus occurs in the 'mother-' or 'grandmother generation'. Sister origins in such cells were found to be colocalized for an entire generation or more, whereas sister origins in slow-growing cells were colocalized for about 0.

A novel regulatory mechanism couples deoxyribonucleotide synthesis and DNA replication in Escherichia coli.

We present evidence for a complex regulatory interplay between the initiation of DNA replication and deoxyribonucleotide synthesis. In Escherichia coli, the ATP-bound DnaA protein initiates chromosomal replication. Upon loading of the beta-clamp subunit (DnaN) of the replicase, DnaA is inactivated as its intrinsic ATPase activity is stimulated by the protein Hda.

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.

Chromosomal replication and the cell membrane.

The importance of the cell membrane in bacterial chromosomal replication continues to emerge. Recent advances include better definition of the biochemical interaction between membrane acidic phospholipids and the replication initiator, DnaA protein, the physiological impact that an altered membrane lipid composition has on chromosomal replication and the identification and characterization of recently identified membrane-associated proteins that regulate replication and participate in chromosomal segregation.

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.

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 chromosome's origin of replication, oriC. Loss of either Fis or IHF perturbs synchronous initiation from oriC copies in rapidly growing E. coli.

Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein.

Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.