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Biomedical subjects

H Echols

Publications and source records attributed to H Echols.

At least 55 records · Page 3Linked to original sources
PubMed1983

Role of the Xis protein of bacteriophage lambda in a specific reactive complex at the attR prophage attachment site.

Phage lambda controls its integration and excision by differential catalysis of the forward and reverse reactions. The lambda Int protein is required for both directions, but Xis for excision only. Previous electron microscopic observations have shown that Int protein forms a stable, condensed protein-DNA complex with the phage (attP) and prophage left (attL) substrate sites, but not with the host (attB) or prophage right (attR) sites. We have found that Int and Xis together produce a stable, condensed complex with attR. The attR complex involves the P region DNA to the left of the crossover point (O site). In contrast, the attP complex includes DNA on both sides of the crossover point (P and P'), and the attL structure involves the P' DNA to the right of O. In the presence of Int and Xis, the attL and attR sites form a paired structure. We conclude that the role of Xis is to provide a distinct reactive structure at attR, allowing attL and attR to pair efficiently.

Bacteriophage lambda↗
PubMed1983

Identification of the epsilon-subunit of Escherichia coli DNA polymerase III holoenzyme as the dnaQ gene product: a fidelity subunit for DNA replication.

Based on extensive genetic and biochemical studies, the multisubunit DNA polymerase III holoenzyme is considered responsible for the chain-elongation stage in replication of the genome of Escherichia coli and is thus expected to be the major determinant of fidelity as well. Previous experiments have shown that two mutations conferring a very high mutation rate on E. coli, mutD5 and dnaQ49, decrease severely the 3' leads to 5' exonucleolytic editing activity of the polymerase III holoenzyme. To identify more precisely the nature of these mutations, we have carried out genetic mapping and complementation experiments. From these studies and experiments by others, we conclude that the most potent general mutator mutations in E. coli occur in a single gene, dnaQ. To define further the role of the dnaQ gene, we have used two-dimensional gel electrophoresis to compare the labeled dnaQ gene product with purified polymerase III holoenzyme. The dnaQ product comigrates with the epsilon-subunit, a 25-kilodalton protein of the polymerase III "core" enzyme. We conclude that the epsilon-subunit of polymerase III holoenzyme has a special role in defining the accuracy of DNA replication, probably through control of the 3' leads to 5' exonuclease activity.

DNA Polymerase III↗
PubMed1983

Mutator strains of Escherichia coli, mutD and dnaQ, with defective exonucleolytic editing by DNA polymerase III holoenzyme.

The closely linked mutD and dnaQ mutations confer a vastly increased mutation rate on Escherichia coli and thus might define a gene with a central role in the fidelity of DNA replication. To look for the biochemical function of the mutD gene product, we have measured the 3' leads to 5' exonucleolytic editing activity of polymerase III holoenzyme from mutD5 and dnaQ49 mutants. The editing activities of the mutant enzymes are defective compared to wild type, as judged by two assays: (i) decreased excision of a terminal mispaired base from a copolymer substrate and (ii) turnover of dTTP to dTMP during replication with a phage G4 DNA template. Thus, the mutD (dnaQ) gene product is likely to control the editing (proofreading) capacity of polymerase III holoenzyme.

Base Sequence↗
PubMed1982

Control of phage lambda development by stability and synthesis of cII protein: role of the viral cIII and host hflA, himA and himD genes.

The cII protein of bacteriophage lambda has a decisive role in the regulatory switch between the lysogenic and lytic pathways of viral development. Recent work has indicated that cII may be the primary control function providing for the initial partition between the two pathways, with other host and viral regulatory genes acting to determine the levels of cII in an infected cell. We have studied the synthesis and stability of cII protein with two experimental systems, phage infection and a cII-producing plasmid. We have found that the stability of cII is controlled by the host hflA and viral cIII genes; hflA protein facilitates degradation of cII, whereas cIII protects cII. The synthesis of cII appears to be under the positive control of the host himA and himD genes. We conclude that posttranscriptional regulation of cII by host and viral genes is critical for the choice of a developmental pathway.

Bacteriophage lambda↗
PubMed1982

Site-specific DNA condensation and pairing mediated by the int protein of bacteriophage lambda.

The int protein of bacteriophage lambda catalyzes the site-specific integrative recombination that inserts lambda DNA into the host chromosome. The attachment site region of lambda DNA required for this reaction spans 230 base pairs and includes four separable binding sites for int protein. We have used the electron microscope to determine the functional consequences of the interaction of int with its multiple binding sites. We find that int condenses a 230-base pair segment of DNA into a compact structure about 14 nm in diameter; the condensed region includes all of the four binding sites for int. Condensed segments will form paired structures between attachments sites. We suggest that a sequential cooperative interaction between bound int molecules provides for a distinct reactive DNA conformation and for pairing between substrate sites.

Bacteriophage lambda↗
PubMed1981

SOS induction and autoregulation of the himA gene for site-specific recombination in Escherichia coli.

The himA gene of EScherichia coli controls the lysogenization of bacteriophage lambda at the level of catalysis of site-specific recombination and expression of the lambda int and cI genes required for lysogenic development. We have analyzed the regulation of himA by two methods: (i) beta-galactosidase synthesis from a lacZ gene inserted into the himA gene and (ii) detection of radioactive HimA protein after fractionation by two-dimensional gel electrophoresis. We find that himA- mutations produce enhanced expression of the himA gene, indicating that HimA protein controls its own synthesis. The himA gene is also induced by treatment of cells with UV or mitomycin C, suggesting control by the inducible DNA repair (SOS) system regulated by the LexA and RecA proteins. Regulation of himA follows the pattern expected for a typical SOS gene: constitutive high expression in mutants that have inactive LexA or the altered RecA conferred by the recA441 (tif1) mutation and low noninducible expression in a mutant that has a deleted recA gene. We conclude that the himA gene is a component of the inducible SoS response, repressed by LexA and induced by the capacity of activated RecA to cleave LexA. We suggest that HimA may be subject to SOS induction because it functions as an "acquisitionase" for new genetic material and thus is of special utility under conditions of impaired capacity for growth of the bacterial population.

Coliphages↗
PubMed1981

Retroregulation of the int gene of bacteriophage lambda: control of translation completion.

Bacteriophage lambda regulates the integration--excision reaction as a crucial aspect of the choice of pathway during lysogenic or lytic viral development. This control involves differential expression of the tightly linked, partially overlapping int and xis genes from two promoter sites: pI, positively regulated by cII/cIII proteins, and pL, positively regulated by N protein. After lambda infection, Int is synthesized from the pI transcript under cII regulation; however, very little Int is produced from the pL RNA because of the existence of a cis-acting regulatory element, sib, on the opposite side of the int gene from the pL promoter. Presumably sib serves to prevent unwanted synthesis of Int protein during the lytic response; the Int protein necessary for excisive recombination from a prophage can be supplied by pL transcription because sib is separated from int by prophage insertion. We have studied the effect of sib on nearby lambda genes by means of gel electrophoresis of labeled proteins from infected cells. Deletion of the sib region greatly enhances production of Int protein without substantial effect on Xis production; thus, sib regulation normally is highly specific for Int. When the sib region is moved past int and xis by deletion, regulation of the adjacent gene for the protein Ea22 occurs, suggesting that sib regulation can work on other genes. Although synthesis of wild-type Int is severely inhibited by sib, shorter Int protein fragments generated by nonsense mutations escape sib regulation, indicating that the regulation is translational and occurs near the completion stage of protein synthesis. Regulation by sib thus exhibits novel regulatory features: distal location, recombinational control, and regulation of the completion of protein synthesis. Because Int and Ea22 control is lost in a RNase III- host, we suggest that sib regulation might involve RNase III cleavage of a RNA duplex region that includes sib and the regulatory target (normally the int gene). We note such a potential site within int.

Bacteriophage lambda↗
PubMed1980

DNA sequence of regulatory region for integration gene of bacteriophage lambda.

The cII and cIII proteins specified by bacteriophage lambda direct the lysogenic response to infection through the coordinate establishment of repression and integration of the viral DNA. The regulatory activity of cII/cIII involves positive regulation of two promoter sites: the p(E) promoter, turning on expression of the cI protein that maintains lysogeny, and the p(I) promoter, activating synthesis of the Int protein for integrative recombination. Regulation of the p(I) promoter provides for differential expression of the Int protein with respect to the excision-specific Xis protein from the closely linked int and xis genes. We have determined the DNA sequence of the p(I) promoter region for wild-type lambda DNA and for two classes of mutations: intc mutations, which result in a high rate of Int synthesis in the absence of cII, and deletion mutations, some of which eliminate cII-activated expression of the int gene. We find a sequence with considerable homology (11 of 15 bases) to a "typical" (computer-generated) promoter sequence, adjacent to a region with striking homology (11 of 14 bases) to part of the p(E) promoter region. This presumed p(I) sequence overlaps the start of the xis gene and includes the site of two intc point mutations. A cII-insensitive xis(+) deletion partially removes the proposed p(I) sequence; a deletion that leaves the p(I) sequence intact but terminates 21 bases upstream does not interfere with cII activation of the int gene. From our results and the analysis of the p(E) region, we suggest that cII acts in the promoter -35 recognition region to facilitate binding by RNA polymerase at the -10 interaction region. Differential expression of the int and xis genes results because the p(I) transcript lacks the initiation codon for Xis protein synthesis.

Bacteriophage lambda↗
PubMed1979

Studies on the binding of lambda Int protein to attachment site DNA: identification of a tight-binding site in the P' region.

We have used three approaches to studying the interaction of lambda Int protein with bacteriophage attachment site DNA, POP': location of binding sites by retention of DNA fragments in a filter binding assay, reconstruction of a binding site by DNA synthesis and protection of a binding site from an exonuclease. Retention of restriction fragments on nitrocellulose filters in the presence of Int protein was used to locate binding sites. A high affinity binding site lies in P' between base pairs -6 and +173 from the center of the common core sequence, and low affinity sites are found in the 200 base pair region left of position -6. Reconstruction of the high affinity binding site region from the right using primed DNA synthesis and testing for filter binding in the presence of Int protein shows that sequences sufficient for tight binding of Int protein lie to the right of position +66. When attachment site DNA is protected by bound Int protein against digestion by exonuclease III, four Int dependent protection bands are seen in positions +58, +68, +79 and +88. This can be interpreted either as showing that four Int protein monomers bind to the high affinity region in series, or as evidence for wrapping of the DNA around Int protein, leading to structural changes resembling those occurring to DNA in nucleosomes.

Bacteriophage lambda↗