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Biomedical subjects

R R Sinden

Publications and source records attributed to R R Sinden.

70 records · Page 4Linked to original sources
PubMed1988

Analysis of trimethylpsoralen photoreactivity to Z-DNA provides a general in vivo assay for Z-DNA: analysis of the hypersensitivity of (GT)n B-Z junctions.

We have described an exonuclease III/photoreversal procedure to map, with base pair resolution, the bases which have photoreacted with 4,5',8-trimethylpsoralen (Me3-psoralen) forming either monoadducts or interstrand cross-links in DNA (20). This assay allows quantitation of relative rates of Me3-psoralen photobinding to bases in DNA at levels as low as one cross-link per 8,000 base pairs. This assay should be useful for a wide variety of applications of Me3-psoralen photobinding to DNA. Here, we demonstrate the applicability of the Me3-psoralen exo III assay for analysis of the conformation of the Z forming sequences (GT)12ATGT and GAATTC(TG)6TA(TG)6. We have shown previously that Me3-psoralen forms crosslinks in the 5'TA within the (CG)6TA(CG)6 sequence when it exists in the B conformation but not when it exists in the Z conformation (34). More recently we have confirmed this result with the exo III assay and have shown at least a hundred fold increase in Me3-psoralen photoreactivity at the 5'AT sequence within the EcoR I sites (GAATTC) which presumably represent B-Z junctions flanking (CG)6TA(CG)6 (20). Here we demonstrate both the characteristic decrease in psoralen photobinding to 5'TAs within (GT)12ATGT and (TG)6TA(TG)6 and the hyperreactivity of B-Z junctions. These characteristic properties of Me3-psoralen photobinding provide an assay for Z-DNA that is applicable in vivo. The general applicability of this approach for assaying Z-DNA in vivo is discussed.

Biotechnology↗
PubMed1987

Supercoiling in prokaryotic and eukaryotic DNA: changes in response to topological perturbation of plasmids in E. coli and SV40 in vitro, in nuclei and in CV-1 cells.

Changes in DNA linking number have been observed in plasmid DNA purified from E. coli cells after the cells were treated with chloroquine. Chloroquine, a DNA intercalating drug, unwinds the DNA, decreasing the levels of negative supercoiling. Following this in vivo topological perturbation, within minutes DNA gyrase decreases DNA linking number producing more negatively supercoiled DNA topoisomers. Following the removal of the drug from cells, within minutes topoisomerase 1 or DNA gyrase increases the linking number restoring the original level of supercoiling. Analogous changes in DNA linking number after addition of chloroquine are observed in purified plasmid DNA, and in purified SV40 minichromosomes in the presence of exogenous topoisomerase. Changes in linking number are also observed in SV40 chromosomes in isolated nuclei and in SV40 DNA purified from CV-1 cells following topological perturbation with chloroquine. These results suggest that eukaryotic cells may have mechanisms to maintain a defined level of DNA supercoiling.

Animals↗
PubMed1987

Reduced 4,5',8-trimethylpsoralen cross-linking of left-handed Z-DNA stabilized by DNA supercoiling.

Z-DNA-forming sequences, (GT)21, (GT)12ATGT, and (CG)6TA(CG)6, were cloned into plasmids. These sequences formed left-handed Z-DNA conformations under torsional tension from negative supercoiling of DNA. 4,5',8-Trimethylpsoralen, on absorption of 360-nm light, forms monoadducts and interstrand cross-links in DNA that exists in the B-helical conformation. Trimethylpsoralen cross-links were introduced into the potential Z-DNA-forming sequences in relaxed DNA when these sequences existed as B-form DNA. In supercoiled DNA when these sequences existed in the Z conformation, the rate of cross-linking was greatly reduced, and trimethylpsoralen did not form monoadducts appreciably to Z-DNA. As an internal control in these experiments, the rates of cross-linking of the Z-DNA-forming sequences were measured relative to that of an adjacent, cloned sequence that could not adopt a Z conformation. The initial relative rates of cross-linking to Z-DNA-forming sequences were dependent on the superhelical density of the DNA, and the rates were ultimately reduced by factors of 10-15 for Z-DNA in highly supercoiled plasmids. This differential rate of cross-linking provides a novel assay for Z-DNA. Initial application of this assay in vivo suggests that a substantial fraction of (CG)6TA(CG)6, which existed as Z-DNA in plasmid molecules purified from cells, existed in the B conformation in vivo.

Base Sequence↗
PubMed1984

Interstrand psoralen cross-links do not introduce appreciable bends in DNA.

Analysis of the X-ray crystallographic structure of an 8-methoxypsoralen-thymine monoadduct has led to the suggestion that psoralen cross-links would bend DNA by as much as 70 degrees [Peckler, S., Graves, B., Kanne, D., Rapoport, H., Hearst, J. E., & Kim, S.-H. (1982) J. Mol. Biol. 162, 157-172]. DNA can exist in a stably bent configuration in solution as recently demonstrated from analysis of polyacrylamide gel electrophoresis and differential decay of birefringence. Using these techniques, we have investigated the structure of DNA cross-linked with 8-methoxypsoralen and 4,5',8-trimethylpsoralen. The results are not consistent with cross-links introducing any appreciable stable bend in double-stranded DNA molecules. Results suggest that photobound 4,5',8-trimethylpsoralen molecules lengthen DNA by the equivalent of about one base pair per photobound adduct. We have also determined that 4,5',8-trimethylpsoralen cross-links are introduced preferentially into 5'-TA compared to 5'-AT DNA sequences.

Base Sequence↗
PubMed1984

Cruciform transitions in DNA.

The rates of transition between the cruciform and linear conformations of a perfectly inverted repeated lac operator DNA sequence have been measured using a trimethylpsoralen intrastrand cross-linking assay. The rate and extent of the linear to cruciform transition were dependent on temperature and on the superhelical density of the DNA. Apparent half-lives for this transition were between 4-9 min at 37 degrees C for supercoiled DNAs as isolated from cells. The half-life for the cruciform to linear transition in relaxed DNA was about 30 s at 37 degrees C. Mg2+ stabilized both conformations but stabilized the linear form to a greater degree than the cruciform. The rates of transition were temperature dependent suggesting enthalpies of activation of 26.3 kcal mol-1 for the cruciform to linear transition and 33.4 kcal mol-1 for the linear to cruciform transition. The rate of the linear to cruciform transition was slower at 50 than 37 degrees C. Heating above 70 degrees C resulted in the loss of the cruciform structure.

Cloning, Molecular↗
PubMed1983

Perfect palindromic lac operator DNA sequence exists as a stable cruciform structure in supercoiled DNA in vitro but not in vivo.

A perfect palindromic 66-base pair (bp) DNA sequence derived from the lac operator and cloned into plasmid pMB9 [Betz, J. L. & Sadler, J. R. (1981) Gene 13, 1-12] can exist in a 66-bp linear form or as two 33-bp cruciform arms. The fraction of the sequence in the cruciform depends on the superhelical density of the plasmid DNA. Relaxed DNA contains no cruciforms. The palindrome in the cruciform structure is cut by EcoRI endonuclease at the base of the cruciform arms, releasing 33-bp fragments; when in the linear form only 66-bp fragments are produced. The cruciform structure is fixed by trimethylpsoralen crosslinks in the cruciform arms. This together with the EcoRI cutting provides an assay for the cruciform structures in the DNA of living cells. Using this assay we show that the cruciform structure rarely if ever exists in vivo, but after DNA isolation greater than 90% of the sequence is in cruciforms. Results suggest that the plasmid DNA as organized in vivo either lacks sufficient torsional tension to form this cruciform or the palindrome is restrained in the linear form by other bound molecules.

Base Sequence↗
PubMed1983

Herpes simplex virus types 1 and 2 induce shutoff of host protein synthesis by different mechanisms in Friend erythroleukemia cells.

Herpes simplex virus type 1 (HSV-1) and HSV-2 disrupt host protein synthesis after viral infection. We have treated both viral types with agents which prevent transcription of the viral genome and used these treated viruses to infect induced Friend erythroleukemia cells. By measuring the changes in globin synthesis after infection, we have determined whether expression of the viral genome precedes the shutoff of host protein synthesis or whether the inhibitor molecule enters the cells as part of the virion. HSV-2-induced shutoff of host protein synthesis was insensitive to the effects of shortwave (254-nm) UV light and actinomycin D. Both of the treatments inhibited HSV-1-induced host protein shutoff. Likewise, treatment of HSV-1 with the cross-linking agent 4,5',8-trimethylpsoralen and longwave (360-nm) UV light prevented HSV-1 from inhibiting cellular protein synthesis. Treatment of HSV-2 with 4,5',8-trimethylpsoralen did not affect the ability of the virus to interfere with host protein synthesis, except at the highest doses of longwave UV light. It was determined that the highest longwave UV dosage damaged the HSV-2 virion as well as cross-linking the viral DNA. The results suggest that HSV-2 uses a virion-associated component to inhibit host protein synthesis and that HSV-1 requires the expression of the viral genome to cause cellular protein synthesis shutoff.

Animals↗
PubMed1982

Organization of herpes simplex virus type 1 deoxyribonucleic acid during replication probed in living cells with 4,5',8-trimethylpsoralen.

The structure of herpes simplex virus type 1 (HSV-1) DNA in the nuclei of living infected cells was studied with the DNA photoaffinity probe 4,5',8-trimethylpsoralen. The rate of photobinding to HSV-1 DNA was compared to that of a suitable internal control at different times during infection. The rates of photobinding to DNA packaged in virions, capsids, and prereplicative and postreplicative DNA were characteristically different. By 4 h after infection, after the initiation of DNA replication, the rate of photobinding to HSV-1 DNA increased 4 times relative to the rate of binding to the host DNA. The enhanced rate of photobinding to HSV-1 DNA was maintained at all later times during infection and was not affected when frequent single-strand breaks were introduced in HSV-1 DNA by gamma irradiation of infected cells. The results suggest that the bulk of the replicating herpes DNA is free of torsional tension and that the differing rates of photobinding are attributable to changes in accessibility of the HSV-1 DNA. The results are compatible with previous proposals, based on in vitro studies, that intranuclear HSV-1 DNA is primarily free of nucleosomal organization and suggest that there are few, if any, unrestrained DNA supercoils averaged over the entire HSV-1 genome.

Affinity Labels↗
PubMed1982

New procedure using a psoralen derivative for analysis of nucleosome associated DNA sequences in chromatin of living cells.

Sites for restriction endonuclease cleavage in double helical DNA are blocked from cleavage when the photoaffinity drug trimethylpsoralen is photobound at or near the site. In general, Hind III sites are about 15 fold more sensitive to inactivation than the other restriction sites which were tested, although sensitivity of different Hind III sites seems to vary somewhat depending on base sequences adjacent to the site. Hind III sites can be inactivated in two ways; one which completely blocks action of the specific restriction endonuclease and one permitting the introduction of a swivel which relaxes DNA supercoiling without producing a double strand break. Nucleosomes and perhaps other protein-DNA complexes can protect the underlying DNA sequence from trimethylpsoralen photobinding and thus protect restriction sites from inactivation. This property can be exploited to determine if specific sites are accessible to the psoralon probe in vivo and thus to establish if specific nucleotide sequences are nucleosome associated. Using this procedure evidence is obtained that nucleosomes on SV40 DNA in living infected cells are either distributed randomly or at many discrete alternate sites that approach a random distribution.

Animals↗
PubMed1981

Chromosomes in living Escherichia coli cells are segregated into domains of supercoiling.

Torsional tension in the DNA double helix can be detected in living cells of Escherichia coli from measurements of the rate of trimethylpsoralen photobinding to the intracellular DNA. Here we show that this tension is relaxed in vivo when single-strand DNA breaks are introduced by gamma-irradiation and that approximately 160 nicks per genome equivalent of DNA are required to relax greater than 95% of the tension. Chromosomes containing less than 160 nicks per genome equivalent lose only a part of the tension, depending on the number of nicks. The remaining tension is maintained during incubations of cells at 0 degrees C. Chromosomes with tension relaxed by incubation of cells with inhibitors of DNA gyrase interact with the trimethylpsoralen probe independently of the number of nicks introduced by gamma-irradiation. The results fit a model in which the chromosome in growing E. coli cells (mean generation time, 30 min) is segregated into 43 +/- 10 domains of supercoiling per genome equivalent of DNA or 120 +/- 30 domains per nucleoid. The number of domains is unchanged in cells depleted of nascent RNA by growth with rifampicin, but varies somewhat in cells growing at different rates in different media.

Chromosomes, Bacterial↗
PubMed1980

Torsional tension in the DNA double helix measured with trimethylpsoralen in living E. coli cells: analogous measurements in insect and human cells.

The rate of covalent photobinding of trimethylpsoralen to DNA is greater when the DNA is wound with negative superhelical tension than when it is relaxed. In vitro the rate of photobinding is directly proportional to the negative superhelical density of the DNA. Thus measurement of the rate of photobinding provides an assay for probing in vivo unrestrained tension in the winding of the DNA double helix. This approach has been applied to measure torsional tension in DNA as it is packaged in living E. coli. Drosophila and HeLa cells. A method is described for measuring the rate of photobinding to intracellular DNA and rRNA, and for using the latter measurement as an internal control of the rate of me3-psoralen photobinding in vivo. This permits more accurate and reproducible measurement of changes in the DNA-psoralen photobinding reaction. The me3-psoralen probe interacts with intracellular bacterial DNA as expected for a purified DNA duplex wound with superhelical density sigma = -0.05 +/- 0.01. This superhelical tension is relaxed in cells when multiple single-strand breaks are introduced into the chromosomal DNA by gamma-irradiation. Similar relaxation occurs when cells are treated with the DNA gyrase inhibitor coumermycin. The results suggest that the DNA double helix is wound with torsional tension in vivo and that DNA supercoils which are equilibrated with this tension are not completely restrained in nucleosome-like structures. Torsional tension in the DNA of eucaryotic cells is not detectable in analogous measurements of the packaged DNA of HeLa and Drosophila cells. The simplest interpretation of this finding is that, within the limits of detection, all superhelical turns in the DNA are restrained in nucleosomes or nucleosome-like structures in these eucaryotic cells.

Animals↗
PubMed1978

Topography and kinetics of genetic recombination in Escherichia coli treated with psoralen and light.

Genetic exchanges appear to be involved in repair of cross-linked DNA. Kinetics for completion of repair and strand rejoining controlled by the recA(+) gene were examined in Escherichia coli treated with psoralen and light. The results suggest the following model for genetic recombination. After cross-linking treatment, cells in a population initiate repair in near synchrony. Removal of DNA cross-links, preparation of substrate for recombination, and initiation of the first recA-dependent event are completed in less than 1 min. Recombination events occur singly in each cell or chromosome, and require 2.3 +/- 0.4 min at 32 degrees for the recA(+)-dependent step. After completion of the first event, subsequent recombination events occur in a sequential or progressive fashion around the chromosome or in clusters which may consist of one or more domains of the folded chromosome. The time required to proceed to successive sites is either a constant, independent of the distance on the chromosome, or is quite small compared to 2.3 min. DNA substrate for recombination decays with approximate first-order kinetics and the rate is dependent on the number of unrepaired sites. Cell survival can be expressed as a competition between completion of all repair events and the simultaneous decay of chromosomes to forms not reparable by recombination.Equations relating kinetics for completion of repair, the size distribution of DNA molecules, and cell survival are derived for the above model, using as parameters only rate constants for recombination and decay of substrate, and number of events per chromosome. An excellent correlation is found between experimentally determined and theoretical values.

DNA Repair↗
PubMed1978

Repair of cross-linked DNA and survival of Escherichia coli treated with psoralen and light: effects of mutations influencing genetic recombination and DNA metabolism.

Repair of cross-linked DNA was studied in Escherichia coli strains carrying mutations affecting DNA metabolism. In wild-type cells, DNA strands cut during cross-link removal were rejoined during a subsequent incubation into high-molecular-weight molecules. This rejoining was dependent on gene products involved in genetic recombination. A close correlation was found relating recombination proficiency, the rate of strand rejoining, and formation of viable progeny after DNA cross-linking by treatment with psoralen and light. Wild-type cells and other mutants which were Rec+ (sbcB, recL, recL sbcB, recB recC sbcA, recB recC sbcB, xthA1, and xthA11) rejoined cut DNA strands at a rate of 0.8 +/- 0.1 min -1 at 37 degrees C and survived 53 to 71 cross-links per chromosome. recB, recC, recB recC, recF, or polA strains showed reduced rates of strand rejoining and survived 4 to 13 cross-links per chromosome. Recombination-deficient strains (recA, recB recC sbcB recF, recB recL) and lexA failed to rejoin DNA strands after crosslink removal and were unable to form colonies after treatments producing as few as one to two cross-links per chromosome. Strand rejoining occurred normally in cells with mutations affecting DNA replication (dnaA, danB, dnaG, and dnaE) under both permissive and nonpermissive conditions for chromosome replication. In a polA polB dnaE strain strand rejoining occurred at 32 degree C but not at 42 degree C, indicating that some DNA synthesis was required for formation of intact recombinant molecules.

DNA Repair↗
PubMed1975

Repair of cross-linked DNA in Escherichia coli.

The repair of DNA containing interstrand cross-links in Escherichia coli was studied by following the temporal sequence of DNA-related metabolic events in cells exposed to psoralen plus light. Mutations in some genes controlling replication, recombination, and repair strongly influence these specific events. Results reported here are consistent with a cross-link repair mechanism involving sequential excision and recombination.

Binding Sites↗