Search PubMed⌕ Search

Biomedical subjects

M Zylicz

Publications and source records attributed to M Zylicz.

54 records · Page 3Linked to original sources
PubMed1988

Initiation of lambda DNA replication reconstituted with purified lambda and Escherichia coli replication proteins.

Using highly purified bacteriophage lambda and E. coli replication proteins, we were able to reconstitute an in vitro system capable of replication ori lambda-containing plasmid DNA. The addition of a new E. coli factor, the grpE gene product, to this replication system reduced the level of dnaK protein required for efficient DNA synthesis by at least 10-fold, and also allowed the isolation of a stable DNA replication intermediate. Based on all available information, we propose a molecular mechanism for the action of the dnaK and grpE proteins during the prepriming reaction leading to lambda DNA synthesis.

Bacterial Proteins↗
PubMed1988

Purification and properties of the NusB protein of Escherichia coli.

Mutations in the nusB gene of Escherichia coli block transcriptional antitermination mediated by the N gene protein of bacteriophage lambda. We describe here two methods of overproducing the NusB protein in E. coli and a method of purifying NusB to apparent homogeneity on a large scale. Purified NusB directly stimulates transcriptional antitermination by the lambda N protein in vitro. It behaves as a monomer (Mr = 15,689) during gel permeation chromatography and gradient sedimentation. The number of NusB molecules in a wild type E. coli K12 cell ranges from about 3,000 to about 6,000 molecules/cell, depending on the growth medium, and is about 50-80% of the number of molecules of the core component of RNA polymerase in the cell. This implies that NusB has a major role in regulating chain elongation during the transcription of E. coli genes. Many E. coli strains with nusB mutations cannot grow at low temperature. However, a sup+ strain with the suppressible amber mutation nusBam115 can grow at 42 degrees C. Since such a strain does not produce NusB protein detectable by immunoprecipitation with anti-NusB, normal amounts of NusB are not essential for the survival of E. coli at 42 degrees C.

Bacterial Proteins↗
PubMed1988

Role of the Escherichia coli DnaK and DnaJ heat shock proteins in the initiation of bacteriophage lambda DNA replication.

We examined the role of two Escherichia coli heat shock proteins, the dnaK and dnaJ gene products, during the initiation of lambda dv DNA replication in vitro. Using 14C-labeled lambda P protein we showed that the DnaK and DnaJ heat shock proteins function together to release lambda P protein from the preprimosomal complex consisting of lambda origin of replication-lambda O-lambda P-DnaB protein. Hydrolysis of ATP, catalyzed presumably by DnaK, is required during this reaction. Substitution of DnaK protein with that of the mutant DnaK756 protein blocks lambda P release. After DnaK and DnaJ action, the preprimosomal complex, isolated on Sepharose 4B, can support lambda dv DNA replication without any additional prepriming proteins. Using DnaK-affinity chromatography we showed that both lambda O and lambda P proteins bind to DnaK protein. The lambda P protein interacts with DnaK protein in a salt-resistant, hydrophobic manner, and ATP hydrolysis is necessary to elute at least part of lambda P protein from the DnaK-affinity column. The proposed mechanism of action of the prokaryotic DnaK and DnaJ heat shock proteins agrees with the hypothesis that Hsp70, the DnaK analogue of eukaryotes, uses ATP to disrupt hydrophobic aggregates [Pelham, H. R. B. (1986) Cell 46, 959-961].

Bacteriophage lambda↗
PubMed1987

The grpE protein of Escherichia coli. Purification and properties.

The grpE gene of Escherichia coli was first identified because a mutation in it, grpE280, prevented bacteriophage lambda DNA replication in vivo. Subsequent work resulted in the identification of the grpE protein in two-dimensional gels and its classification as a heat shock protein. Here we report the purification of the grpE protein. We show that overproduction of grpE occurs in dnaK 103 bacteria which do not produce a functional Mr 72,000 dnaK protein. The grpE protein was purified from this strain primarily by its specific retention on a dnaK affinity column. The interaction between these two proteins, which is stable in the presence of 2 M KCl, allowed other proteins to be washed from this column. grpE was then eluted by ATP, which disrupts the interaction. During purification, grpE activity was monitored by its ability to complement an in vitro lambda dv DNA replication system dependent on the lambda O and lambda P proteins. The effect of ATP on the dnaK-grpE complex was also observed during sedimentation of the two proteins in glycerol gradients. Purified grpE protein has a Mr of approximately 23,000 under both denaturing and native conditions, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation, respectively. However, in the presence of dnaK under native conditions, grpE cosediments with dnaK. When ATP is added to the gradient, the complex is disrupted, and the two proteins sediment independently as monomers.

Adenosine Triphosphate↗
PubMed1987

Enzymology of the pre-priming steps in lambda dv DNA replication in vitro.

We have examined some of the early pre-priming steps of bacteriophage lambda dv DNA replication in vitro. Previous experiments have shown that bacteriophage lambda replication requires host RNA polymerase-dependent RNA synthesis near or at the origin of replication (ori lambda) to initiate DNA synthesis. Using a crude Fraction II enzymatic system we have shown that during RNA polymerase action, at least the bacteriophage lambda O and lambda P replication proteins as well as the host dnaB protein must be present to initiate ori lambda-specific DNA replication. The presence of three other host initiation proteins, dnaG primase, dnaJ, and dnaK, is not required during RNA polymerase action. Because of the apparent absence of a requirement for the dnaJ and dnaK pre-priming proteins during the transcriptional activation step, we propose that the early events of lambda dv DNA replication, prior to action by the dnaG primase, can be divided into two recognizable steps: an early step which requires at least RNA polymerase, lambda O, lambda P, and dnaB, and a subsequent step which requires the action of at least the dnaJ and dnaK proteins.

Bacterial Proteins↗
PubMed1986

The nucleotide sequence of the Escherichia coli K12 dnaJ+ gene. A gene that encodes a heat shock protein.

The Escherichia coli dnaJ gene product is required for bacteriophage lambda DNA replication at all temperatures. It is also essential for bacterial viability in at least some conditions, since mutations in it result in temperature-sensitive bacterial growth. We have previously cloned the dnaJ gene and shown that its product migrates as a Mr 37,000 polypeptide under denaturing conditions. Here we present the primary DNA sequence of the dnaJ gene. It codes for a processed basic protein (63 basic and 51 acidic amino acids) composed of 375 amino acids totaling Mr 40,973. The predicted NH2-terminal amino acid sequence, overall amino acid composition, and isoelectric point agree well with those of the purified protein. We present evidence that the rate of expression of the dnaJ protein is increased by heat shock under the control of the htpR (rpoH) gene product.

Amino Acid Sequence↗
PubMed1986

Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both lambda DNA replication at all temperatures and host growth at high temperature.

We have identified the grpE gene product as the B25.3 heat shock protein of Escherichia coli on the following evidence: (i) a protein similar in size and isoelectric point to B25.3 was induced after infection of UV-irradiated bacteria by lambda grpE+ transducing phage, (ii) mutant phage lambda grpE40, isolated by its inability to propagate on grpE280 bacteria, failed to induce the synthesis of the B25.3 protein, and (iii) lambda grpE+ revertants, derived from phage grpE40 as able to propagate on grpE280 bacteria, simultaneously recovered the ability to induce synthesis of the B25.3 protein. In addition, we show that E. coli bacteria carrying the grpE280 mutation are temperature sensitive for bacterial growth at 43.5 degrees C. Through transductional analysis and temperature reversion experiments, it was demonstrated that the grpE280 mutation is responsible for both the inability of lambda to replicate at any temperature tested and the lack of colony formation at high temperature. At the nonpermissive temperature the rates of synthesis of DNA and RNA were reduced in grpE280 bacteria.

Bacterial Proteins↗
PubMed1986

Escherichia coli DnaK protein possesses a 5'-nucleotidase activity that is inhibited by AppppA.

AppppA and the DnaK protein have both been hypothesized to function in regulating the heat shock response of Escherichia coli. The proposals are that AppppA serves as a signal (alarmone) to turn on the heat shock response, whereas the DnaK protein is necessary to turn off the heat shock response. A simple model would be that the DnaK protein turns off the response by degrading AppppA. We disproved this model by demonstrating that the DnaK protein possesses a 5'-nucleotidase activity capable of degrading many cellular nucleotides but not AppppA. Although AppppA was not a substrate, it did inhibit the 5'-nucleotidase activity of the DnaK protein. This inhibition may be specific and have biological function since the mutant DnaK756 protein, which is defective in turning off the heat shock response, is partially desensitized to AppppA inhibition. These findings led us to consider other possible mechanisms for AppppA and the DnaK protein in heat shock regulation.

5'-Nucleotidase↗
PubMed1985

Purification and properties of the dnaJ replication protein of Escherichia coli.

The Escherichia coli dnaJ gene was originally discovered because mutations in it blocked bacteriophage lambda DNA replication. Some of these mutations were subsequently shown to interfere with bacterial growth at high temperature, suggesting that dnaJ is an essential protein for the host as well. The first step in purifying the dnaJ protein was to overproduce it at least 50-fold by subcloning its gene into the pMOB45 runaway plasmid. The second step was the development of an in vitro system to assay for its activity. A Fraction II extract from dnaJ259 mutant bacteria was shown to be unable to replicate lambda dv DNA unless supplemented with an exogenous source of wild-type dnaJ protein. Using this complementation assay we purified the dnaJ protein to homogeneity from the membrane fraction of an overproducing strain of bacteria. The purified dnaJ protein was shown to be a basic (pI 8.5), yet hydrophobic, protein of Mr 37,000 and 76,000 under denaturing and native conditions, respectively, and to exhibit affinity for both single- and double-stranded DNA. Using a partially purified lambda dv replication system dependent on the presence of the lambda O and P initiator proteins and at least the host dnaB, dnaG, dnaJ, dnaK, single-stranded DNA-binding protein, gyrase, RNA polymerase holoenzyme, and DNA polymerase III holoenzyme, we have shown that the dnaJ protein is required at a very early step in the DNA replication process.

Amino Acids↗
PubMed1985

Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli.

Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the lambda O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.

Bacterial Proteins↗
PubMed1984

Purification and properties of the Escherichia coli dnaK replication protein.

The Escherichia coli dnaK+ gene was cloned into the "runaway" plasmid vector pMOB45 resulting in a large overproduction of the dnaK protein. The dnaK protein was purified by following its ability to complement the replication of single-stranded M13 bacteriophage DNA in a reaction system dependent on the presence of the lambda O and P DNA replication proteins. The DNA replication activity of the dnaK protein is also essential for lambda dv DNA replication in vitro, since antibodies against it were shown to inhibit the reaction. Purified dnaK protein preparations possess a weak ATPase activity and an autophosphorylating activity which copurify with its DNA replication activity throughout all purification steps. The dnaK protein is an acidic largely monomeric protein of Mr = 72,000 and 78,400 under denaturing and native conditions, respectively. The amino acid composition and N-terminal amino acid sequence match those predicted from the DNA sequence of the dnaK gene (Bardwell, J.C.A., and Craig, E. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 848-852).

Amino Acid Sequence↗
PubMed1984

Bacteriophage lambda replication proteins: formation of a mixed oligomer and binding to the origin of lambda DNA.

The purified bacteriophage lambda replication proteins O and P sediment separately in metrizamide gradients of low ionic strength as dimers. Together they interact with each other forming an oligomer, composed of two molecules of lambda O and one molecule of lambda P. The lambda O-P oligomer is active in the in vitro replication of ori lambda-containing DNA. Equilibrium sedimentation in preformed metrizamide density gradients under conditions that separate DNA-protein complexes from free proteins was employed in order to study possible interactions among the lambda replication proteins and ori lambda DNA. It was found that the lambda P protein binds specifically to ori lambda-containing plasmid DNA only in the presence of lambda O protein. About 100 molecules of lambda O and 10 molecules of lambda P form a complex with the ori lambda DNA. The lambda DNA-lambda O-lambda P complex was shown to be active in an in vitro replication system. Since the physical interactions between ori lambda and lambda O and between lambda P and the Escherichia coli dnaB replication protein are well documented, the evidence for a lambda O-P interaction presented in this paper provides the missing link in the molecular mechanism that enables lambda to direct the host replication machinery to the replication of its own DNA.

Bacteriophage lambda↗
PubMed1983

Escherichia coli dnaJ- and dnaK-gene products: synthesis in minicells and membrane-affinity.

Escherichia coli dnaJ- and dnaK-gene products have been identified in a system of minicells infected with the appropriate transducing lambda phages. The molecular weights of these polypeptides in dodecyl sulphate/acrylamide electrophoresis amounted to 39,000 and 77,000, respectively. Equilibrium sedimentation of minicell lysates in metrizamide density gradients has revealed that both these host proteins, indispensable for lambda DNA replication, are membrane-bound.

Bacterial Proteins↗
PubMed1983

The dnaK protein modulates the heat-shock response of Escherichia coli.

E. coli bacteria respond to a sudden upward shift in temperature by transiently overproducing a small subset of their proteins, one of which is the product of the dnaK gene. Mutations in dnaK have been previously shown to affect both DNA and RNA synthesis in E. coli. Bacteria carrying the dnaK756 mutation fail to turn off the heat-shock response at 43 degrees C. Instead, they continue to synthesize the heat-shock proteins in large amounts and underproduce other proteins. Both reversion and P1 transduction analyses have shown that the failure to turn off the heat-shock response is the result of the dnaK756 mutation. In addition, bacteria that overproduce the dnaK protein at all temperatures undergo a drastically reduced heat-shock response at high temperature. We conclude that the dnaK protein is an inhibitor of the heat-shock response in E. coli.

Bacterial Proteins↗
PubMed1983

The dnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system.

The Escherichia coli dnaK gene product, originally defined by mutations that blocked lambda phage DNA replication, is known to be necessary for E. coli viability. We have purified dnaK protein to homogeneity and have demonstrated that it possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi. The proof that this ATPase activity is encoded by the dnaK+ gene relies primarily on the fact that the dnaK756 mutation results in the production of an ATPase activity with altered physical properties. The dnaK protein is phosphorylated in vitro and in vivo, probably as a result of an autophosphorylation reaction. The lambda O and P replication proteins were shown to interact in vitro with the dnaK protein. The ATPase activity of the dnaK protein was inhibited by purified lambda P protein and stimulated by purified lambda O protein. Moreover, the dnaK protein participates in the initiation of DNA synthesis in an in vitro DNA replication system that is dependent on the O and P proteins. Anti-dnaK protein immunoglobulin specifically inhibited DNA synthesis in this system.

Adenosine Triphosphatases↗
PubMed1981

Interactions between phage lambda replication proteins, lambda DNA and minicell membrane.

Gentle methods for minicell lysis and lysate fractionation have been elaborated: lysis by T4 lysozyme without detergents, and fractionation by equilibrium sedimentation in a metrizamide density gradient, both at low ionic strength. In the lysates of phage-lambda-infected minicells the lambda DNA, trapped at a prereplicative step [Witkiewicz, H. and Taylor, K. (1979) Biochim. Biophys. Acta 564, 31-36], appeared in two peaks of different buoyant densities: as a membrane-bound and a free lambda DNA. The covalently-closed-circular form of lambda DNA appeared exclusively in the membrane fraction. The lambda-coded proteins, synthesized in lambda-infected minicells, appeared in two major fractions: as membrane-bound and as free proteins, and in one minor fraction, bound with free lambda DNA. Neither lambda protein engaged in the initiation of DNA replication was present in the fraction of free proteins: the P-gene product was membrane-associated, and the O-gene product formed a complex with free lambda DNA. The effect of high ionic strength (KCl) and of detergents (Triton X-100 and sarcosyl) on the binding of replication proteins with lambda DNA and with the membrane was studied. The non-ionic detergent, Triton X-100 caused displacement of a part of lambda DNA from the membrane to the free lambda DNA peak; both lambda replication proteins were bound with free lambda DNA. The binding of the O protein with lambda DNA was relatively stable, but was destroyed by the ionic detergent, sarcosyl.

Bacteriophage lambda↗