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R Thresher

Publications and source records attributed to R Thresher.

11 recordsLinked to original sources
PubMed1997

MutS mediates heteroduplex loop formation by a translocation mechanism.

Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction.

Adenosine Triphosphatases↗
PubMed1993

Replicatively active complexes of DnaA protein and the Escherichia coli chromosomal origin observed in the electron microscope.

DnaA protein and the Escherichia coli chromosomal origin (oriC) form an initial complex at an early stage in the initiation of DNA replication. We have used electron microscopy to determine which structure among the several formed in the reconstitution of this multicomponent system is the replicatively active complex. One distinctive structure could be correlated with activity and localized to oriC, whilst several others could not. Formation of an open complex in the next stage of initiation was accompanied by the presence of a structure similar in size and shape to that of the functional initial complex. Whereas the initial complex was observed with either ATP or the ADP-forms of DnaA protein, only the ATP-form was effective in producing the open complex. Mutagenesis of several DNA sequence elements in oriC, known to be important for replication, was employed to determine the effects of these alterations on formation of the initial complex. As judged by electron microscopy and by functional assays, the region containing the four 9-mer dnaA boxes proved to be essential for the formation of the initial complex, while the three contiguous AT-rich 13-mers, known sites for opening of oriC, were not.

Adenosine Diphosphate↗
PubMed1992

Characterization of DNA-binding and strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding protein.

Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms, including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 M(r) heterotrimer containing 70,000, 36,000 and 14,000 M(r) subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (K omega greater than 10(9) M-1) and co-operativity (omega = 10,000 to 100,000). Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sepl and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA, y-RPA stimulated the strand-exchange activity of Sepl approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sepl occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sepl mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast.

DNA, Single-Stranded↗
PubMed1992

Electron microscopic study of (A)BC excinuclease. DNA is sharply bent in the UvrB-DNA complex.

Nucleotide excision repair in Escherichia coli is initiated by the UvrA, UvrB and UvrC proteins. UvrA is the damage recognition subunit, makes an A2B1 complex with the targeting subunit UvrB, and the complex binds to the lesion site; UvrA dissociates leaving behind a very stable UvrB-DNA complex that is recognized by the trigger subunit, UvrC, and the ensuing UvrB-UvrC heterodimer makes two incisions, one on either side of the lesion. Using electron microscopy, we investigated the structures of these early A, A-B intermediates on DNA containing ultraviolet light photoproducts. UvrA, which is known to bind to DNA as a dimer and produce a DNase I footprint of 33 base-pairs does not change the trajectory of DNA appreciably. The A2B1 complex clearly shows a bipartite structure and its effect on the trajectory of the DNA was not consistently straight or kinked. In contrast, the DNA in the preincision UvrB-DNA complex appears to be severely kinked; 43% of the molecules are bent by 80 degrees or more, with an average bending angle of 127 degrees. It appears that protein-induced bending is an important step on the pathway leading to excision of the damaged nucleotide by (A)BC excinuclease.

Adenosine Triphosphatases↗
PubMed1989

Gap formation is associated with methyl-directed mismatch correction under conditions of restricted DNA synthesis.

A covalently closed, circular heteroduplex containing a G-T mismatch and a single hemimethylated d(GATC) site is subject to efficient methyl-directed mismatch correction in Escherichia coli extracts when repair DNA synthesis is severely restricted by limiting the concentration of exogenously supplied deoxyribonucleoside-5'-triphosphates or by supplementing reactions with chain-terminating 2',3'-dideoxynucleoside triphosphates. However, repair under these conditions results in formation of a single-strand gap in the region of the molecule containing the mismatch and the d(GATC) site. These findings indicate that repair DNA synthesis required for methyl-directed correction can initiate in the vicinity of the mispair, and they are most consistent with a repair reaction involving 3'----5' excision (or strand displacement) from the d(GATC) site followed by 5'----3' repair DNA synthesis initiating in the vicinity of the mismatch.

DNA Repair↗
PubMed1985

Endothelial cell repopulation after intracameral acetic acid irrigation.

A significant, but transient corneal edema followed inadvertent intracameral irrigation of acetic acid in two patients. Since postsurgical corneal edema generally indicates corneal endothelial dysfunction, the effects of acetic acid on corneal endothelium were studied in the cat where the endothelial regenerative capacity is thought to be comparable to that of humans. The anterior chambers of feline eyes were irrigated with 0.25% acetic acid or balanced salt solution in a double-masked manner. Marked corneal opacification and thickening were evident following exposure to the acetic acid but did not occur following irrigation with balanced salt solution alone. Following intracameral acetic acid irrigation, a few endothelial cells were found in the anterior chamber angle but could not be found on Descemet's membrane. Over an 8-week period gradual repopulation of Descemet's membrane by endothelial cells occurred, first in the periphery and later in the central cornea, coincident with resolution of corneal edema.

Acetates↗
PubMed1984

Open-angle glaucoma associated with posterior polymorphous dystrophy. A clinicopathologic study.

Clinical and histopathologic features of a 62-year-old black man with posterior polymorphous dystrophy and open-angle glaucoma requiring trabeculectomy are reported. The surgical procedure was modified to allow en bloc resection of the deep limbal tissue and peripheral iris. Light and electron microscopy of the specimen revealed a high insertion of the iris into the posterior portion of the trabecular meshwork with compression of the intertrabecular spaces. These findings are similar to changes that have been reported in primary congenital glaucoma and suggest a developmental anomaly of the anterior chamber angle in this form of glaucoma associated with posterior polymorphous dystrophy.

Cornea↗
PubMed1983

Temporary use of silicone oil in the treatment of proliferative vitreoretinopathy. An experimental study with a new animal model.

Homologous fibroblasts were injected into vitrectomized rabbit eyes. This injection produced a total retinal detachment in all ten eyes of a control series. In each case the detachment was due to the contraction of a membrane which had grown on the retinal surface as seen in human proliferative vitreoretinopathy (PVR). In another group of ten eyes, silicone oil was injected before the fibroblastic membrane contracted, and removed again a few weeks later. This temporary tamponade of silicone oil reduced the rate of detachment to 50%. Silicone oil did not prevent the preretinal membrane from growing but mechanically decreased its contraction.

Animals↗
PubMed1982

The clearance of intravitreal triamcinolone acetonide.

Since high corticosteroid levels are important in achieving inhibition of intraocular cellular proliferation, we studied the clearance of an intravitreally injected slowly dissolving corticosteroid in the rabbit eye. Triamcinolone acetonide (0.5 mg) disappeared rapidly in eyes that underwent a combined vitrectomy and lensectomy (average, 6.5 days) and more slowly in eyes that underwent vitrectomy only (average, 16.8 days) compared with unoperated normal rabbit eyes (average, 41 days). The ophthalmoscopic disappearance of the white crystals correlated well with a sensitive colorimetric test for clearance of the corticosteroid. Direct observation is therefore an accurate method of assessing the absorption of the corticosteroid. If used therapeutically, this study suggests that more frequent injections of triamcinolone acetonide would be necessary in eyes that underwent vitrectomy, compared with normal eyes.

Animals↗
PubMedDate not supplied

Axenfeld-Rieger syndrome. A spectrum of developmental disorders.

The clinical and histopathologic features of Axenfeld's anomaly and Rieger's anomaly and syndrome are reviewed, and recent findings regarding the pathogenesis of this spectrum of developmental disorders are discussed. Based on these observations, it has been suggested that a developmental arrest, in the third trimester of gestation, of tissues derived from the neural crest cells accounts for the ocular and most of the nonocular abnormalities in this group of disorders. Since previous collective terms, such as mesodermal dysgenesis and anterior chamber cleavage syndrome, are not consistent with these new observations, the alternative name, Axenfeld-Rieger syndrome, has been proposed. The differential diagnosis of the syndrome includes two additional spectra of disorders: the iridocorneal endothelial syndrome and the posterior polymorphous dystrophies. The most serious ocular problem in Axenfeld-Rieger syndrome is the associated glaucoma, which occurs in a high percentage of patients and is typically difficult to control. Recent observations regarding the mechanism of the glaucoma, as reviewed in this paper, provide guidance in the management of this aspect of Axenfeld-Rieger syndrome.

Abnormalities, Multiple↗