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L D Kosturko

Publications and source records attributed to L D Kosturko.

14 recordsLinked to original sources
PubMed1999

Sequence-specific cleavage by bacteriophage T4 endonuclease II in vitro.

The 136 codon (408 bp) denA gene encoding endonuclease II (Endoll) of bacteriophage T4 was unambiguously identified through sequencing and subsequent cloning. Endoll prepared from cloned DNA through coupled in vitro transcription-translation nicked and cut DNA in vitro in a sequence-specific manner. In vitro (and in vivo), the bottom strand was nicked between the first and second base pair to the right of a top-strand CCGC motif shared by favoured in vitro and in vivo cleavage sites; top-strand cleavage positions varied. To the right of the cleavage position, favoured in vitro sites lacked a sequence element conserved at favoured in vivo sites. In pBR322 DNA, the sites cleaved in vivo as previously described were also cleaved in vitro, but in vitro additional sites were nicked or cleaved and the preference for individual sites was different. Also, different from the in vivo reaction, nicking was more frequent than ds cutting; in many copies of a ds cleavage site, only the bottom strand was nicked in vitro. A model is discussed in which sequential nicking of the two strands, and different factors influencing bottom-strand nicking and top-strand nicking, can explain the differences between the in vitro and the in vivo reaction.

Bacteriophage T4↗
PubMed1998

Endonuclease II of coliphage T4: a recombinase disguised as a restriction endonuclease?

EndoII shares with restriction endonucleases the property of cleaving foreign DNA while leaving the endonuclease-encoding genome intact, ensuring the survival of one DNA species in the cell. In addition, in vivo EndoII cleaves a specific DNA sequence and cleavage is context dependent. These context effects extend over at least 1000 bp, largely limiting cleavage to once within this distance. Like homing endonucleases, in vivo EndoII recognizes a long, asymmetric and degenerate consensus sequence which has two distinct parts. Recognition of one part of the consensus sequence involves base-specific bonds, and recognition of the other involves sequence-dependent helical structure. EndoII fulfills an obvious short-term survival role in ensuring the dominance of phage DNA in an infected cell, but may also have a long-term evolutionary role, producing gene-size fragments of foreign DNA to be enrolled in the phage genetic repertoire.

Bacteriophage T4↗
PubMed1996

Short-range and long-range context effects on coliphage T4 endonuclease II-dependent restriction.

Synthetic sites inserted into a plasmid were used to analyze the sequence requirements for in vivo DNA cleavage dependent on bacteriophage T4 endonuclease II. A 16-bp variable sequence surrounding the cleavage site was sufficient for cleavage, although context both within and around this sequence influenced cleavage efficiency. The most efficiently cleaved sites matched the sequence CGRCCGCNTTGGCNGC, in which the strongly conserved bases to the left were essential for cleavage. The less-conserved bases in the center and in the right half determined cleavage efficiency in a manner not directly correlated with the apparent base preference at each position; a sequence carrying, in each of the 16 positions, the base most preferred in natural sites in pBR322 was cleaved infrequently. This, along with the effects of substitutions at one or two of the less-conserved positions, suggests that several combinations of bases can fulfill the requirements for recognition of the right part of this sequence. The replacements that improve cleavage frequency are predicted to influence helical twist and roll, suggesting that recognition of sequence-dependent DNA structure and recognition of specific bases are both important. Upon introduction of a synthetic site, cleavage at natural sites within 800 to 1,500 bp from the synthetic site was significantly reduced. This suggests that the enzyme may engage more DNA than its cleavage site and cleaves the best site within this region. Cleavage frequency at sites which do not conform closely to the consensus is, therefore, highly context dependent. Models and possible biological implications of these findings are discussed.

Bacteriophage T4↗
PubMed1994

Multiple molecules of integration host factor (IHF) at a single DNA binding site, the bacteriophage lambda cos I1 site.

Integration host factor (IHF) is an E coli protein that binds DNA sequence-specifically and serves as a cofactor in many intracellular processes including lambda DNA packaging. In gel shift experiments, cos DNA, a DNA fragment containing the recognition signal for lambda DNA packaging, forms multiple protein-DNA complexes when combined with pure IHF. Copper(II)-1,10 orthophenanthroline footprinting of individual IHF-cos DNA complexes shows that multiple complex formation does not result from IHF binding to successive sites on the cos DNA fragment. Instead, the footprinting of DNA from two IHF-cos complexes shows protection at one site alone. DNA in the first complex is only partially protected from nucleolytic cleavage, while DNA in the second, slower-moving, complex is completely protected at the same binding site. Quantitative Western blotting experiments determined the relative stoichiometry of IHF to DNA in the two complexes. The results confirm that two molecules of IHF bind at a single site in the cos fragment. This site, cos I1, has two matches to the IHF consensus sequence, but the two matches overlap by eight of thirteen nucleotides. A search of the DNA sequence around cos, using an expanded IHF consensus sequence, has revealed additional, low-affinity consensus matches, contiguous to these. The extent of the copper(II)-1,10 orthophenanthroline footprint and the stoichiometry of the IHF-cos I1 complexes suggest that either two molecules of IHF bind to overlapping sites, or IHF binds to a site of low affinity contiguous to a strong site. Application of a thermodynamic model to the results of gel shift experiments with IHF and cos DNA suggests that multiple complex formation requires cooperative interaction between the two IHF binding sites.

Autoradiography↗
PubMed1993

DNA determinants of restriction. Bacteriophage T4 endonuclease II-dependent cleavage of plasmid DNA in vivo.

Endonuclease II of coliphage T4 is necessary for the in vivo restriction of plasmid DNA in phage-infected cells. Double-stranded restriction cleavage at 12 sites in pBR322 commenced before 10-min postinfection with T4 at 37 degrees C and proceeded more slowly in the presence of competing phage DNA than in its absence, utilizing the same sites in both cases; in a 200-base pair segment of the plasmid, single-stranded nicks also were frequent. The plasmid sites were cleaved with a speed that varied with the site, yielding frequencies of cleavage at different sites varying between 10 and 90%, at 50-min postinfection. All sites contained good matches to a consensus, 5'-GRCCGCNTYGC-3', most frequently cleaved around the variable central base pair, generating fragments with blunt ends or 1-2-base 5' overhangs. Using the frequency of cleavage to determine a weighted consensus, a larger sequence, 5'-CGRCCGCNTTGSYNGC-3', was identified. Thus, DNA sequence elements 3' to the cut site appear important for rapid cleavage. Several models describing the sequence-dependent structure of DNA suggest structural anomalies around the cleavage sites. The endonuclease II restriction system is most similar to type II systems, although it differs from known type II systems in several respects.

Amino Acid Sequence↗
PubMed1989

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.

Bacterial Proteins↗
PubMed1986

In vitro encapsidation of plasmid DNA into human adenovirus empty capsids.

Plasmid DNA, added to extracts of human adenovirus type 3-infected HeLa cells, binds to empty viral capsids and can be purified using cesium density gradient centrifugation. The fraction of DNA bound depends on the amount of DNA added to the extract, and the capsid partially protects the bound DNA from digestion by DNase I. This capsid binding of plasmid DNA does not require the presence of the adenovirus DNA packaging sequence. However, the presence of the adenovirus packaging sequence in the plasmid results in better protection of the bound plasmid molecule from cellular nucleases.

Adenoviruses, Human↗
PubMed1982

Polar encapsidation of adenovirus DNA: cloning and DNA sequence of the left end of adenovirus type 3.

The left-end adenovirus type 3 DNA sequence is very similar to those of other subgroup B adenoviruses, especially in the area between the HinfI site (320 base pairs) and the early-region Ia gene. This segment of the genome has been implicated as necessary for the left-end polarity of adenovirus DNA encapsidation. This segment and the sequences flanking it are compared with the corresponding sequences of adenovirus type 5 and adenovirus type 12, and the extent and pattern of intersubgroup homologies are discussed.

Adenoviruses, Human↗
PubMed1979

Selective repression of transcription by base sequence specific synthetic polymers.

We report the effect of novel synthetic polymers on deoxyribonucleic acid (DNA) directed ribonucleic acid (RNA) synthesis in vitro. Polymers contained base-selective monomers, including a GC-specific phenazine derivative and an AT-specific triphenylmethane dye. Radical chain polymerization was carried out in aqueous solution by using monomers bound to a template DNA, which was obtained from either lambda or T7 bacteriophage. Polymers were isolated and reannealed with DNA samples, including competitive mixtures of T7 and lambda DNAs. We measured transcription from DNA-polymer complexes by using Escherichia coli RNA polymerase and determined not only the reduction in total transcription levels but also the relative inhibition of lambda- or T7-specific transcription by using a hybridization assay. The results show that micromolar concentrations of individual dyes are sufficient to cause substantial inhibition of transcription when the dyes are incorporated into polymers. More significantly, a number of the polymers inhibited more strongly transcription from the DNA which had served as template for polymer synthesis than from the DNA present as competitor in the annealing process. We conclude that template synthesis of DNA-binding polymers can lead to preferential inhibition of function of the original template. The apparent relative affinity of polymer for competing DNAs can be altered by at least an order of magnitude depending on which DNA was used as the synthesis template. The results offer a new approach to improving the specificity of DNA-binding drugs.

Coloring Agents↗
PubMed1979

Structure of DNA within three isometric bacteriophages.

This paper describes a model for the structure of DNA contained in three morphologically similar bacteriophages--T7, P22 and phiCd-1--based on the transient electric dichroism of intact phage. The reduced dichroism of each of the phages at perfect orientation is within the range +0.12 to +0.19. Assuming that the phage orientation axis is that which passes from the apex through the tail, the measured dichroism suggests that DNA is wrapped in closely packed, co-axial solenoids with the axis of the solenoids tipped 43.5 degrees +/- 2.5 degrees from the orientation axis of the phage. All three phages show a large permanent dipole moment, with respective values of 5600, 200,000 and 500,000 Debye for T7, phiCd-1 and P22. The radius of the equivalent sphere for the three phages calculated from the rotational relaxation time for the rise of dichroism is in agreement with birefringence and electron microscope observations. The circular dichroism spectra of all three bacteriophages indicate that the local DNA helicity is similar in each case.

Coliphages↗
PubMed1976

Late events in T4 bacteriophage production. I. Late DNA replication is primarily exponential.

The possibility of a switch in the mechanism of T4 DNA replication, from an exponential-bidirectional mode at early stages to a nonexponential (rolling circle) mode at later stages of phage development, has been investigated. The conclusion that DNA replication does not involve such a change in mechanism for the majority of replicating molecules is based on the analysis of the clonal distribution of mutants specifically induced at late times after infection. The clonal distribution of mutants, induced by adding 5-bromodeoxyuridine to infected cells at a time when 100 phage equivalents of DNA had accumulated, fits the pattern predicted by exponential replication.

Bromodeoxyuridine↗
PubMed1972

In vivo production of an RNA-DNA copolymer after infection of Escherichia coli by bacteriophage T4.

An RNA-DNA copolymer was isolated from Escherichia coli infected with bacteriophage T4. The RNA and DNA are covalently linked, and in the same polynucleotide strand. The DNA of the copolymer hybridizes specifically to the left strand of phage T4 DNA. The copolymer is produced in cells infected with amber mutants of phage T4 deficient in DNA replication and is not inhibited by the addition of chloramphenicol.

Bromodeoxyuridine↗