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A Kornberg

Publications and source records attributed to A Kornberg.

At least 19 recordsLinked to original sources
PubMed1992

Opening of the replication origin of Escherichia coli by DnaA protein with protein HU or IHF.

Opening of the three tandem repeats of a 13-mer in the replication origin (oriC) of Escherichia coli is a prime event in the replication in vitro of minichromosomes (Bramhill, D., and Kornberg, A. (1988) Cell 54, 915-918). DnaA, the initiator protein, requires protein HU or IHF, along with a millimolar level of ATP and negative superhelical density in the plasmid to open this region. The extent of opening, as judged by cleavage by a single-strand-specific endonuclease (i.e. P1 nuclease), correlated closely with replication of the oriC plasmid. In an initial complex, preceding opening of the 13-mers, the footprint of DnaA protein bound by ATP covered its four 9-mer recognition sequences. The footprint of the nucleotide-free form of the protein, by contrast, was more extensive and thus, less specific.

Bacterial Proteins↗
PubMed1992

Opposed actions of regulatory proteins, DnaA and IciA, in opening the replication origin of Escherichia coli.

The opening of the three tandem 13-mers (iterons) in the replication origin (oriC) of Escherichia coli by DnaA protein, assisted by protein HU or IHF (Hwang, D. S., and Kornberg, A. (1992) J. Biol. Chem. 267, 23083-23086), represents an essential early stage in the initiation of chromosomal replication (Bramhill, D., and Kornberg, A. (1988) Cell 54, 915-918). We now show by mutational alterations of the 13-mer region that oriC function, both in vitro and in vivo, requires AT-richness in the left 13-mer and sequence specificity in the middle and right 13-mers. Interactions of DnaA protein with the middle and right 13-mers are crucial for the opening of the region. Binding of the protein to the top strand of the 13-mers appeared to maintain single-strandedness in the bottom strand. IciA protein, the inhibitor of initiation, binds the three 13-mers and blocks the opening of the region. The degrees of inhibition by IciA protein of 13-mer opening and of oriC plasmid replication observed with mutant forms of the 13-mers could be correlated with the binding affinity of IciA protein. Whereas the binding of IciA protein to the 13-mers did not affect the binding of DnaA protein to its four 9-mers boxes, interaction of DnaA protein with the 13-mers was blocked. The selective interactions of DnaA and IciA proteins with the 13-mer region appear to be components of the on/off switch that controls initiation of E. coli chromosomal replication.

Bacterial Proteins↗
PubMed1992

The polyphosphate kinase gene of Escherichia coli. Isolation and sequence of the ppk gene and membrane location of the protein.

Polyphosphate kinase (PPK) catalyzes the reversible transfer of the terminal phosphate of ATP to form a long-chain polyphosphate (polyP) (Ahn, K., and Kornberg, A. (1990) J. Biol. Chem. 265, 11734-11739). The Escherichia coli gene (ppk) encoding PPK has been cloned, sequenced, and overexpressed (about 100-fold). The gene possesses an open reading frame for 687 amino acids (mass of 80,278 Da). PPK has been purified from overproducing cells after release from attachment to the cell outer membrane; the purified soluble PPK reassociate with cell membrane fractions. About 850 molecules of PPK are found in a wild type cell.

Amino Acid Sequence↗
PubMed1992

The chromosome origin of Escherichia coli stabilizes DnaA protein during rejuvenation by phospholipids.

DnaA protein (the initiator protein) binds and clusters at the four DnaA boxes of the Escherichia coli chromosomal origin (oriC) to promote the strand opening for DNA replication. DnaA protein activity depends on the tight binding of ATP; the ADP form of DnaA protein, generated by hydrolysis of the bound ATP, is inactive. Rejuvenation of ADP-DnaA protein, by replacement with ATP, is catalyzed by acidic phospholipids in a highly fluid bilayer. We find that interaction of DnaA protein with oriC DNA is needed to stabilize DnaA protein during this rejuvenation process. Whereas DnaA protein bound to oriC DNA responds to phospholipids, free DnaA protein is inactivated by phospholipids and then fails to bind oriC. Furthermore, oriC DNA facilitates the high affinity binding of ATP to DnaA protein during treatment with phospholipids. A significant portion of the DnaA protein associated with oriC DNA can be replaced by the ADP form of the protein, suggesting that all of the DnaA protein bound to oriC DNA need not be rejuvenated between rounds of replication.

Adenosine Diphosphate↗
PubMed1992

Plasma crosslinked fibrin polymers: quantitation based on tissue plasminogen activator conversion to D-dimer and measurement in normal and patients with acute thrombotic disorders.

Plasma crosslinked fibrin polymers (XLFP) are formed as a result of in vivo hemostatic activation and are elevated in thrombotic disease. We have investigated the plasmic degradation of plasma XLFP in vitro to provide information regarding the pattern of crosslinking and the composition of degradation products. Plasma XLFP were identified by sodium dodecyl sulfate (SDS)-agarose electrophoresis and Western blotting and quantitated by gel scanning. D-dimer was measured by enzyme-linked immunosorbent assay and the results were verified by SDS-polyacrylamide gel electrophoresis and Western blotting of the digests. Complete degradation of XLFP occurred only after supplementation of plasma with plasminogen (5 U/mL) and incubation with recombinant tissue plasminogen activator (rt-PA), indicating that the normal plasma plasminogen concentration limits plasmic degradation in vitro. Gel electrophoresis showed that the principal terminal degradation products of XLDP were fragments D, DD, and E, indicating that crosslinking occurred primarily through gamma chain dimers. After adding a low concentration of thrombin to plasma in vitro, XLFP increased progressively before clotting, and the concentration correlated with the increase in the D-dimer concentration after degradation (r = .98). Plasma XLFP and D-dimer concentrations in plasmic digests were significantly elevated in patients with stroke (150 +/- 83 micrograms/mL and 88 +/- 32 micrograms/mL), myocardial infarction (217 +/- 110 micrograms/mL and 84 +/- 30 micrograms/mL), and venous thrombosis (187 +/- 80 micrograms/mL and 86 +/- 19 micrograms/mL) compared with normals (28 +/- 12 micrograms/mL and 25 +/- 7 micrograms/mL). There was a strong correlation between the plasma concentration of XLFP and the D-dimer immunoreactivity of plasma after plasmic degradation (r = .87). The results indicate that XLFP in plasma are crosslinked primarily through gamma chains and degrade to fragment DD with plasminogen activation. Also, the immunoreactivity of in vitro plasmic digests of plasma reflects the concentration of XLFP and may provide a useful indirect measure of in vivo hemostatic activation in patients with thrombotic disease.

Acute Disease↗
PubMed1992

Prereplicative complexes of components of DNA polymerase III holoenzyme of Escherichia coli.

Stepwise reconstitution of the subunits of DNA polymerase III holoenzyme of Escherichia coli offers insights into the organization and function of this multisubunit assembly. A highly processive, holoenzyme-like activity can be generated when the gamma complex, in the presence of ATP and a primed template, activates the beta subunit to form a preinitiation complex, and this is then followed by addition of the core polymerase. Further analysis of early replicative complexes has now revealed: 1) that the gamma complex can stably bind a single-stranded DNA binding protein (SSB)-coated template, 2) that neither SSB coating of the template nor a proper primer terminus is required to form the preinitiation complex, and 3) that the gamma complex stabilizes the preinitiation complex in the presence of ATP and destabilizes it in the presence of adenosine 5'-O-(thiotriphosphate). Based on these findings, a sequence of stages can be formulated for an activation of the beta subunit that enables it to bind the template-primer and thereby interact with the core to create a processive polymerase.

Adenosine Triphosphate↗
PubMed1992

Features of replication fork blockage by the Escherichia coli terminus-binding protein.

Blockage of the progress of a DNA replication fork in Escherichia coli can be ascribed to an inhibition of helicase action at the orientation-specific binding of a termination sequence (ter) by the ter-binding protein (Lee, E.H., Kornberg, A., Hidaka, M., Kobayashi, T., and Horiuchi, T. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9104-9108). These observations have been extended to include the PriA helicase, thus confirming that blockage is general for helicases. The site of arrest of synthesis by a replication fork is at the very first nucleotide of the 22-base pair E. coli-terB sequence. Strand displacement by DNA polymerases is also inhibited, but is less profound and is orientation-specific. The ter sequences of plasmids R1-terR and -terL and of plasmids R6K and R100 have been compared with those of E. coli-terA and -terB.

Adenosine Triphosphatases↗
PubMed1992

IciA protein, a specific inhibitor of initiation of Escherichia coli chromosomal replication.

Specific binding of IciA protein to the 13-mers in the origin of a minichromosome (oriC) inhibits initiation of replication in vitro by blocking the opening of this region effected by the initiator DnaA protein (Hwang, D.S., and Kornberg, A. (1990) Cell 63, 325-331). Isolation of the iciA gene (Thöny, B., Hwang, D.S., Fradkin, L., and Kornberg, A. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4066-4070) has made possible the construction of an IciA-overproducing strain, which in turn has simplified the isolation of a large quantity of the protein, indistinguishable from that of the wild-type strain. Based on sedimentation and gel filtration, the IciA protein is an elongated dimer of a 33.4-kDa subunit. The specific binding of IciA protein to the 13-mers was stable for 2 h at 30 degrees C. The amounts of IciA protein, detected by immunoassays, increased 4-fold compared with levels (about 100 dimers) in log-phase cells whereas levels of DnaA protein decreased upon entry of cells into the stationary phase.

Bacterial Proteins↗
PubMed1991

Fine balance in the regulation of DnaB helicase by DnaC protein in replication in Escherichia coli.

The DnaC protein of Escherichia coli is essential for replication in vivo and in vitro. In the initiation of replication of a minichromosome at its origin, DnaC delivers the DnaB helicase from a DnaB.DnaC complex to the future replication fork and then departs. However, if an excess of DnaC was present in subsequent steps, it severely inhibited replication by slowing the DnaB helicase at the replication fork. When DnaB was present at a level equimolar with the excess DnaC, the inhibition was relieved, implying that the ratio of DnaC to DnaB is critical for achieving optimal replication activity and avoiding inhibition by DnaC. In vivo, overproduction of DnaC slowed cell growth. This slowing was alleviated by overproducing DnaB at the same time. E. coli strains with a dnaCts gene defective in chromosomal initiation were complemented by the wild-type gene in trans. On the other hand, strains with an elongation-defective dnaCts gene were not complemented by the wild-type dnaC gene. The dominance of the mutant protein suggests that it remains tightly complexed with DnaB at the replication fork, inhibiting elongation even in the presence of the wild-type DnaC.

Alleles↗
PubMed1991

The priB gene encoding the primosomal replication n protein of Escherichia coli.

The gene encoding protein n of the Escherichia coli primosome has been discovered in the rpsF-rpsR-rplI ribosomal protein operon and designated priB. The low copy number of PriB protein and the distinctive codon usage of its gene argue against its being a ribosomal protein. A strain which overproduces PriB was constructed and has been used to purify the protein to homogeneity. The overproduced protein behaves like that purified from wild-type cells.

Amino Acid Sequence↗
PubMed1991

iciA, an Escherichia coli gene encoding a specific inhibitor of chromosomal initiation of replication in vitro.

The gene encoding the protein that binds the three 13-mers in the origin (oriC) of Escherichia coli to block initiation of replication in vitro has been cloned, sequenced, and overexpressed. The gene possesses an open reading frame for 297 amino acids (mass of 33,471 Da). The protein has a motif for DNA-binding (helix-turn-helix) and has homology to a diverse set of prokaryotic regulatory proteins, known as the LysR family. The protein, previously referred to as the 33-kDa protein, has been named IciA (for inhibitor of chromosome initiation). The iciA gene is at 62.8 min on the chromosomal map. Cells with enhanced levels of the protein grow at a normal rate but generally exhibit a pronounced lag upon transfer to a fresh medium.

Amino Acid Sequence↗
PubMed1991

Replication deficiencies in priA mutants of Escherichia coli lacking the primosomal replication n' protein.

The priA gene of Escherichia coli encodes the protein that initiates assembly of the promosome, the entity essential for the replication of phage phi X174 and ColE1-like plasmids in vitro. We have prepared a null priA mutant to assess its role in vivo in replication of phages, plasmids, and the host chromosome. Extracts of this mutant are inert in the initial conversion of the phi X174 viral strand to the duplex form, confirming the absence of the PriA activity. In vivo, the priA mutant fails to produce phi X174 phage and, remarkably, is unable to maintain plasmids that depend on the E. coli chromosome origin as well as those of ColE1. Deficiencies in cell growth and cell division are also manifest.

Adenosine Triphosphatases↗
PubMed1990

Aggregated dnaA protein is dissociated and activated for DNA replication by phospholipase or dnaK protein.

dnaA protein isolated from Escherichia coli is equally distributed between a monomeric form, which is active for initiation of DNA replication, and an inactive, aggregated form which contains phospholipids. Replication activity of the aggregated form can be restored by treatments with either dnaK protein or phospholipase A2. Dissociation of the aggregate by dnaK protein is driven by ATP hydrolysis; action by phospholipase A2 requires a minute concentration of ATP only to stabilize the dissociated protein. Conversion of inactive dnaA-phospholipid complexes to the active form may contribute to the regulation of the initiation of chromosomal replication in E. coli.

Adenosine Triphosphate↗