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G M Weinstock

Publications and source records attributed to G M Weinstock.

At least 91 records · Page 5Linked to original sources
PubMed1984

Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions.

A recA-lacZ protein fusion was constructed in vivo by using bacteriophage Mu dII301(Ap lac). The fusion contained the promoter and first 47 codons of the recA mutant, as determined by DNA sequence analysis. The fusion was cloned and used to construct a recA-lacZ operon fusion at the same site within the recA gene. These fusions were introduced into the Escherichia coli chromosome at the lambda attachment site either as complete or cryptic lambda prophages. Synthesis of beta-galactosidase from these fusions was inducible by UV radiation. As the UV dose was increased, induction became slower and persisted for a longer period of time. At low doses of UV radiation, more beta-galactosidase was produced in a uvrA mutant than in a wild-type strain; however, at high doses, no induced synthesis of beta-galactosidase occurred in a uvrA mutant. recA+ strains carrying either the protein or operon fusion on a multicopy plasmid showed reduced survival after UV irradiation. This UV sensitivity was not exhibited by strains containing a single copy of either fusion, however; hence, the fusions provide a reliable measure of recA expression.

Bacterial Proteins↗
PubMed1983

Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase.

We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.

Antigens, Bacterial↗
PubMed1981

Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Steady state kinetic analysis of ATP hydrolysis.

The DNA-dependent ATPase activity of the recA protein of Escherichia coli shows a complex dependence on ATP concentration. With a single-stranded (SS) DNA cofactor, the Hill coefficient for ATP is 3.3 at pH 8.1 and 1.4 at pH 6.2. With a double-stranded (DS) DNA cofactor, the Hill coefficient is 3.3 at pH 6.2 (no reaction is detectable at pH 8.1). In the presence of SS DNA, the Km for ATP is 20 microM, independent of pH, while with DS DNA at pH 6.2, KmATP is 100 microM. These and other observations indicate that the interaction of recA protein with ATP is influenced by both pH and DNA cofactor. ADP, UTP, dTTP, and GTP are competitive inhibitors of the ATPase activity of recA protein, indicating that there is a single binding site for nucleoside triphosphates. Nucleoside triphosphates, but not ADP, reduce the Hill coefficient for ATP hydrolysis and thus can contribute to the cooperative effect of ATP.

Adenosine Triphosphatases↗
PubMed1981

Interaction of the recA protein of Escherichia coli with adenosine 5'-O-(3-thiotriphosphate).

Incubation of the recA protein of Escherichia coli with the ATP analog adenosine 5'-O-(3-thiotriphosphate) (ATP(gamma S)) in the presence of DNA produces an irreversible inhibition of ATPase activity, although in the presence of ATP, ATP(gamma S) shows an initial competitive inhibition. ATP(gamma S) is not appreciably hydrolyzed by recA protein and the inhibition of ATPase activity is due to the formation of stable complexes which contain equimolar amounts of ATP(gamma S) and recA protein. Formation of stable complexes requires DNA, which is also stably bound to recA protein in the presence of ATP(gammaS), at a ratio of 5 to 10 nucleotides/recA protein monomer. The DNA requirement is satisfied by either single-or double-stranded DNA, and in the latter case, the pH dependence is comparable to that observed for ATP hydrolysis. Binding of ATP(gamma S) is inhibited by other nucleoside di- and triphosphates with efficiencies corresponding to their inhibitory effects on the ATPase activity of recA protein.

Adenosine Triphosphatases↗
PubMed1981

Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Characterization of ATP hydrolysis.

Both single- and double-stranded DNA stimulate the hydrolysis of ATP catalyzed by the recA protein of Escherichia coli. However, the reactions differ in their pH optima, response to recA protein concentration, salt sensitivity, and degree of inhibition by ADP, all of which reflect different requirements for the prehydrolytic binding of single- and double-stranded DNA by the RecA protein. Single- and double-stranded DNA stimulate hydrolysis of the same nucleoside triphosphates, principally (r,d)ATP and (r,d)UTP, suggesting that a single hydrolytic site is utilized in both single- and double-stranded DNA-dependent reactions. recA protein also catalyzes detectable ATP hydrolysis in the absence of exogenous DNA, although the rate is reduced 2 to 3 orders of magnitude. This DNA-independent hydrolysis shows the same nucleotide specificity at pH 6.2 and 7.5, although the rate of hydrolysis depends upon the pH.

Adenosine Triphosphate↗
PubMed1981

Binding of the recA protein of Escherichia coli to single- and double-stranded DNA.

The recA protein of Escherichia coli binds both single- (SS) and double-stranded (DS) DNA; however, the optimal conditions differ for interaction with these DNA substrates. Binding of DS DNA by recA protein is pH dependent (optimum near pH 6.2) and requires a nucleoside triphosphate (ATP) and divalent cation. Substitution of the 5'-O-3'-thiotriphosphate (ATP(gamma S)) for ATP leads to formation of stable complexes of recA protein and DNA that dissociate very slowly. Formation of these complexes is extremely sensitive to ionic strength and pH. However, once formed, the complexes resist changes in pH and high salt concentrations. SS DNA binds to recA protein in the absence of a nucleoside triphosphate, but recA protein-SS DNA complexes are stabilized by ATP(gamma S). At high recA protein/DNA ratios (1 recA protein monomer/30 nucleotides), these complexes sediment in sucrose gradients as large protein-DNA aggregates. Although ATP(gamma S) blocks dissociation of recA protein from DNA, ATP stimulates the release of recA protein from SS DNA. Hydrolysis of the ATP is not required for dissociation since it is also enhanced by ADP and certain nucleoside triphosphates that are not hydrolyzed by recA protein. recA protein binds with different affinities to ribohomopolymers and deoxyhomopolymers. It preferentially binds polydeoxythymidylate and polydeoxycytidylate but does not bind short oligonucleotides, indicating that there is a minimum size requirement for the binding step. The recA protein exists as a heterogeneous aggregate at pH 7.5 and at low ionic strength. At pH 6.2 in the presence of Mg2+, the protein sediments homogeneously as a dimer. At pH 6.2, ATP or ATP(gamma S) promotes an oligomerization of the recA protein which can be observed as filamentous structures by electron microscopy. Oligomerization is not induced by UTP, a nucleoside triphosphate that is efficiently hydrolyzed by the recA protein, but fails to stimulate efficiently recA protein-promoted annealing and assimilation of single-stranded DNA.

Adenosine Triphosphate↗
PubMed1981

Hydrolysis of nucleoside triphosphates catalyzed by the recA protein of Escherichia coli. Hydrolysis of UTP.

Hydrolysis of UTP catalyzed by the recA protein of Escherichia coli is stimulated by both double- (DS) and single-stranded (SS) DNA. DS DNA-dependent UTPase activity has a sharp optimum near pH 6. SS DNA-dependent UTP hydrolysis also is optimal near pH 6, although considerable activity remains at pH 8. Both SS and DS DNA-dependent UTPase activities are nonlinearly dependent on recA protein concentration at pH 6 but the SS DNA-dependent reaction shows a linear dependence on enzyme concentration at pH 8. The Km for UTP in the SS DNA-dependent reaction decreases from 147 microM at pH 8 to 33 microM at pH 6. The Km for UTP in the DS DNA-dependent reaction is 247 microM at pH 6. In addition, the Hill coefficient for UTP in the SS DNA-dependent reaction decreases from 3.5 at pH 8 to 1.9 at pH 6, while in the DS DNA-dependent reaction, the Hill coefficient is 2.4 at pH 6. Thus, the UTPase activity of the recA protein differs from the ATPase activity of recA protein primarily in the pH dependence of KmUTP, Vmax, and response to enzyme concentration of the SS DNA-dependent hydrolysis reaction. These differences may be related to the inability of UTP to substitute effectively for ATP in recA protein-promoted annealing and assimilation of SS DNA.

Bacterial Proteins↗
PubMed1981

tif-1 mutation alters polynucleotide recognition by the recA protein of Escherichia coli.

The requirements for polynucleotide-dependent hydrolysis of ATP and for proteolytic cleavage of phage lambda repressor have been examined for both the wild-type (recA+ protein) and the tif-1 mutant form [tif(recA) protein] of the recA gene product. The recA+ and tif(recA) proteins catalyze both reactions in the presence of long single-stranded DNAs or certain deoxyhomopolymers. However, short oligonucleotides [(dT)12, (dA)14] stimulate neither the protease nor the ATPase activities of the recA+ protein. In contrast, these short oligonucleotides activate tif(recA) protein to cleave lambda repressor without stimulating its ATPase activity. Moreover, both the ATPase and protease activities of the tif(recA) protein are stimulated by poly(rU) and poly(rC) whereas the recA+ protein does not respond to these ribopolymers. We have purified the recA protein from a strain in which the tif mutation is intragenically suppressed. This mutant protein (recA629) is inactive in the presence of (dT)12, (dA)14, poly(rU), and poly(rC) for lambda repressor cleavage and ATP hydrolysis. These results argue that the tif-1 mutation (or mutations) alters the DNA binding site of the recA protein. We suggest that in vivo the tif(recA) protein is activated for cleaving repressors of SOS genes by complex formation with short single-stranded regions or gaps that normally occur near the growing fork of replicating chromosomes and are too short for activating the recA+ enzyme. This mechanism can account for the expression of SOS functions in the absence of DNA damage in tif mutant strains.

Adenosine Triphosphate↗
PubMed1980

recA protein-catalyzed strand assimilation: stimulation by Escherichia coli single-stranded DNA-binding protein.

The single-stranded DNA-binding protein of Escherichia coli significantly alters the strand assimilation reaction catalyzed by recA protein [McEntee, K., Weinstock, G. M. & Lehman, I. R. (1979) Proc. Natl. Acad. Sci. USA 76, 2615--2619]. The binding protein (i) increases the rate and extent of strand assimilation into homologous duplex DNA, (ii) enhances the formation of a complex between recA protein and duplex DNA in the presence of homologous or heterologous single-stranded DNA, (iii) reduces the rate and extent of ATP hydrolysis catalyzed by recA protein in the presence of single-stranded DNA, (iv) reduces the high concentration of recA protein required for strand assimilation, and (v) permits detection of strand assimilation in the presence of the ATP analog, adenosine 5'-O-(O-thiotriphosphate). Single-stranded DNA-binding protein purified from a binding protein mutant (lexC) is considerably less effective than wild-type binding protein in stimulating strand assimilation, a result which suggests that single-stranded DNA-binding protein participates in general recombination in vivo.

Adenosine Triphosphatases↗
PubMed1980

Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule.

A general method for efficiently mutagenizing a predetermined segment of a closed circular duplex DNA molecule was used to construct mutations in two specific regions of the beta-lactamase (bla) gene carried by the small plasmid pBR322. The principle of segment-directed mutagenesis is the use of a single-stranded homologous DNA fragment to direct the nicking of circular duplex DNA within a segment defined by the DNA fragment in a two-step reaction. First, Escherichia coli recA protein is used to catalyze assimilation of the homologous single-stranded DNA, producing a displacement loop ("D-loop") in the circular DNA. Second, a small amount of the single-strand-specific S1 nuclease is used to nick the displaced DNA. The segment-directed nicks are converted to small gaps, which are then mutagenized specifically with sodium bisulfite. A short (128-base pair) restriction endonuclease fragment from the center of the bla gene was used to direct mutagenesis with the result that 7.5% of the recovered plasmids were bla- mutants and 49/51 of these mutants, mapped genetically, were found to lie in a deletion interval whose endpoints approximate those of the restriction fragment. Similar results were obtained when another short fragment covering the beginning of the gene was used; many of these mutations map in the region coding the "signal" sequence thought to be involved in secretion of beta-lactamase.

Base Sequence↗
PubMed1979

ATP-dependent renaturation of DNA catalyzed by the recA protein of Escherichia coli.

The product of the recA gene of Escherichia coli has been purified to near-homogeneity by a simple three-step procedure. Incubation of the recA protein with complementary single strands of DNA, Mg2+, and ATP results in the rapid formation of large DNA aggregates containing many branched structures. As judged by resistance to S1 nuclease and by electron microscopy, these aggregates contain both duplex and single-stranded regions. The renaturation and aggregation of DNA catalyzed by the recA protein is coupled to the hydrolysis of ATP. The recA protein purified from a cold-sensitive recA mutant does not catalyze DNA renaturation or aggregation at 28 degrees C, but does so at 37 degrees C, a finding which correlates with the recombination defect observed in vivo and indicates that this activity is an intrinsic function of the recA protein. These results suggest that the recA protein plays a specific role in strand transfer during recombination and possibly in postreplication repair of damaged DNA.

Adenosine Triphosphate↗
PubMed1979

Initiation of general recombination catalyzed in vitro by the recA protein of Escherichia coli.

Homogeneous recA protein catalyzes the hybridization of single-stranded DNA to homologous regions in duplex DNA. The products are D-loops, which are formed with equal efficiency in linear and supercoiled molecules. This assimilation reaction can be separated into two partial reactions. In the first, recA protein binds to duplex DNA and produces a reA protein-DNA complex. The binding shows a sigmoidal dependence on recA protein concentration, requires ATP, GTP or the gamma-thio analog of ATP, and Mg2+, but does not require hydrolysis of the nucleoside triphosphate. In the second reaction, single-stranded regions of the recA protein-ATP-duplex DNA intermediate hybridize with free complementary single strands to produce D-loop structures. This reaction is coupled to ATP hydrolysis and is analogous to the renaturation of single-stranded DNA catalyzed by the recA protein [Weinstrock, G.M., McEntee, K. & Lehman, I.R. (1979) Proc. Natl. Acad. Sci. USA 76, 126-130]. Hydrolysis of ATP appears to be required in these reactions for dissociation of recA protein from the DNA.

Adenosine Triphosphate↗
PubMed1979

Regional specificity of illegitimate recombination by the translocatable ampicillin-resistance element Tn1 in the genome of phage P22.

Insertions of the translocatable ampicillin-resistance element Tn1 were selected in the genome of the temperate Salmonella phage P22 by growing the phage on hosts carrying the resistance plasmid RP4. Insertions of Tn1 into phage P22 are rare (10(-10) per phage) and nonrandomly distributed in the P22 genome. They are found mainly in the vicinity of the P22 ant gene. Insertions within the ant gene are found at many (at least 15) genetically separable sites, are found equally frequently in both orientations and cause irreversible loss of gene function. Some insertions in ant appear to be associated with an adjecent deletion. Prophage deletions were derived from P22::Tn1 phages by two methods. Low multiplicity transductants have nonrandomly distributed endpoints. One end is at or very near the site of the Tn1 insertion, and the other is in the vicinity of gene 12; however, there are many genetically distinguishable endpoints within gene 12. Prophage deletions selected as survivors of induction of a P22Ap mnt-ts lysogen have similarly nonrandom endpoints, with the Tn1-distal end frequently near the ant gene, as well as gene 12. Physical analysis of several prophage deletions suggests that the Tn1 is intact to the resolution of DNA electron microscopy and that the deletions begin at the end of the Tn1 insertion. These results suggest that illegitimate recombination associated with Tn1 shows regional specificity (i.e., preference for some large areas of the P22 genome over other areas), but that within these regions is quite nonspecific.

Ampicillin↗