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Biomedical subjects

B E Kaplan

Publications and source records attributed to B E Kaplan.

At least 19 recordsLinked to original sources
PubMed2000

Arginine side-chain dynamics in the HIV-1 rev-RRE complex.

The binding of human immunodeficiency virus type 1 (HIV-1) Rev protein to its viral RNA target, stem-loop IIB (SLIIB) within the Rev Response element (RRE), mediates the export of singly-spliced and unspliced viral mRNA from the nucleus to the cytoplasm of infected cells; this Rev-mediated transport of viral RNA is absolutely required for the replication of infectious virus. To identify important features that influence the binding affinity and specificity of this Rev-RRE interaction, we have characterized the arginine side-chain dynamics of the Rev arginine-rich motif (ARM) while bound to a 34 nt RNA oligomer that corresponds to SLIIB. As the specificity of the Rev-RRE interaction varies with salt concentration, arginine side-chain dynamics were characterized at two different salt conditions. Following NMR measurements of (15)N spin relaxation parameters for the arginine (15)N(epsilon) nuclei, the dynamics of the corresponding N(epsilon)-H(epsilon) bond vectors were interpreted in terms of Lipari-Szabo model-free parameters using anisotropic expressions for the spectral density functions. Results from these analyses indicate that a number of arginine side-chains display a surprising degree of conformational freedom when bound to RNA, and that arginine residues having known importance for specific RRE recognition show striking differences in side-chain mobility. The (15)N relaxation measurements at different salt conditions suggest that the previously reported increase in Rev-RRE specificity at elevated salt concentrations is likely due to reduced affinity of non-specific Rev-RNA interactions. The observed dynamical behavior of the arginine side-chains at this protein-RNA interface likely plays an important role in the specificity and affinity of Rev-SLIIB complex formation.

Amino Acid Motifs↗
PubMed1999

Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway.

Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) pyrimidine dimer photoproducts in DNA. Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified. Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA glycosylases have also been characterized. Recently, a novel UV damage endonuclease (UVDE) was identified that initiates an excision repair pathway different from previously established repair mechanisms. Homologues of UVDE have been found in eukaryotes as well as in bacteria. In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimidine dimers and (6-4) photoproducts for the characterization of this UV damage repair pathway. After introduction of single-strand breaks at the 5' sides of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins). FEN-1 homologues from humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions. T4 endonuclease V-incised DNA was processed in the same way. Both nicked and flapped DNA substrates with photolesions (the latter may be intermediates in DNA polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1. The data suggest that the two enzymatic activities, UVDE and FEN-1, are part of an alternative excision repair pathway for repair of UV photoproducts.

Base Sequence↗
PubMed1998

Solid-phase synthesis and characterization of carcinoembryonic antigen (CEA) domains.

Carcinoembryonic antigen (CEA), a 180,000 dalton cell surface glycoprotein expressed on tumors of the colon, breast, ovary, and lung, has seven predicted immunoglobulin-like domains (N-A1-B1-A2-B2-A3-B3), most of which are recognized by distinct monoclonal antibodies. To study the individual domains, we have prepared several of the domains (N, A3, B3, and A3-B3) by solid-phase peptide synthesis. The syntheses were performed by the Fmoc method using single couplings, elevated temperatures for both the coupling and deblocking reactions, and a flexible solvent system for the coupling reactions. The syntheses were accomplished on an in-house built synthesizer which allowed for temperature control and flexible solvent control during the course of the coupling reactions. Due to the large size of the peptides (84-184 residues), it was anticipated that the overall purity of the final product would not exceed 60% even for an average coupling yield of 99.5%. Therefore, several of the peptides were synthesized with a His6 "tail" at the amino terminus, allowing for purification on a Ni-NTA chelate column. For the most part, the purified peptides exhibited single sharp peaks by RP-HPLC, migrated at their expected molecular weights by gel permeation chromatography, gave correct masses by electrospray ionization or matrix-assisted laser desorption ionization time of flight mass spectrometry, gave the expected amino acid analyses, N-terminal sequences, and tryptic maps, and bound their appropriate monoclonal antibodies. The N-domain was extremely hydrophobic, requiring 6M guanidinium hydrochloride for solubilization, the A3 domain was soluble in dilute acid, and the B3 domain had an intermediate solubility. The affinity constants of the A3 domain and several mutants (also made by peptide synthesis) are reported, along with characterization of the 178 amino acid two-domain peptide, A3-B3. Although there is no evidence for proper folding of these domains by NMR, their ability to bind monoclonal antibodies with high affinity suggests that this is a plausible approach for producing individual domains of CEA.

Amino Acid Sequence↗
PubMed1996

Inhibition of transcription factor binding by ultraviolet-induced pyrimidine dimers.

The formation of DNA photoproducts by ultraviolet (UV) light is responsible for the induction of mutations and the development of skin cancer. Cis-syn cyclobutane pyrimidine dimers (pyrimidine dimers) are the most frequent lesions produced in DNA by UV irradiation. Besides being mutagenic, pyrimidine dimers may interfere with other important DNA-dependent processes. To analyze the effects of pyrimidine dimers on the ability of DNA sequences to be recognized by trans-acting factors, we have incorporated site-specific T-T dimers into oligonucleotides containing the recognition sequences of the sequence-specific transcription factors E2F, NF-Y, AP-1, NF kappa B, and p53. In each case, presence of the photodimer strongly inhibited binding of the respective transcription factor complex. Reduction of binding varied between 11- and 60-fold. The results indicate that the most common UV-induced DNA lesion can interfere severely with binding of several important cell cycle regulatory and DNA damage responsive transcription factors. We suggest that inhibition of transcription factor binding may be a major biological effect of UV radiation since promoter regions are known to be repaired inefficiently and since UV damage can deregulate the function of a large number of different factors.

Binding Sites↗
PubMed1994

Topography of a binding site for small amnestic peptides deduced from structure-activity studies: relation to amnestic effect of amyloid beta protein.

Four peptides homologous to amyloid beta protein containing the Val-Phe-Phe (VFF) sequence administered intracerebroventricularly after training caused amnesia for footshock active avoidance training in mice. Results with VFF and other peptides containing VFF or portions thereof were used to generate a topographic map for a hypothetical binding surface for amnestic peptides, termed Z. Effects on retention of footshock active avoidance training were rationalized in terms of fit to Z, making possible design of potential memory-modulating peptidic and nonpeptidic substances. Three peptides that neither improved nor impaired retention blocked the amnestic effects of beta-(12-28), a peptide homologous to amyloid beta protein, opening the way to development of substances that can antagonize the neurotoxic effects of amyloid beta protein on neural structures and thus attenuate symptoms and progression of Alzheimer disease.

Amino Acid Sequence↗
PubMed1994

Polystyrene reverse-phase ion-pair chromatography of chimeric ribozymes.

The use of a reverse-phase polystyrene resin for ion-pair HPLC purification of large amounts of synthetic chimeric DNA-RNA oligomers that is faster and more reliable than previously used techniques has been developed. The preparation of synthetic oligomers containing RNA requires the use of tetrabutylammonium fluoride in the final step, the cleavage of the tert-butyl-dimethyl silyl protecting group from the ribonucleotides. Cleavage is accompanied by the serendipitous formation of ion pairs between tetrabutylammonium cations and the oligomer phosphates. The formation of these ion pairs retards the elution of the oligomer during HPLC, which allows rapid removal of excess tetrabutylammonium fluoride and the concomitant purification of chimeric ribozymes. This technique is based on a correlation between the length of ion-paired oligomers and their retardation during HPLC. The advantages of reverse-phase ion-pair HPLC on polystyrene resin for the fast purification of oligoribonucleotides are discussed and illustrated through the examples of synthesized chimeric ribozymes.

Base Sequence↗
PubMed1993

Ribozyme-mediated inhibition of bcr-abl gene expression in a Philadelphia chromosome-positive cell line.

The bcr-abl fusion gene is the molecular counterpart of the Philadelphia chromosome (Ph1) and is directly involved in the pathogenesis of Ph1+ leukemia. Inhibition of bcr-abl gene expression may have profound effects on the cell biology of Ph1+ cells, as recent experiments with antisense oligonucleotides have shown. In this study we have designed and synthesized a unique ribozyme that is directed against bcr-abl mRNA. The ribozyme cleaved bcr-abl mRNA in a cell-free in vitro system. A DNA-RNA hybrid ribozyme was then incorporated into a liposome vector and transfected into EM-2 cells, a cell line derived from a patient with blast crisis of chronic myelogenous leukemia. The ribozyme decreased levels of detectable bcr-abl mRNA in these cells, inhibited expression of the bcr-abl gene product, p210bcr-abl, and inhibited cell growth. This anti-bcr-abl ribozyme may be a useful tool to study the cell biology of Ph1+ leukemia and may ultimately have therapeutic potential in treating patients with Ph1 leukemias.

Base Sequence↗
PubMed1993

Incorporation of nonbase residues into synthetic oligonucleotides and their use in the PCR.

Oligonucleotides containing the nonbase residues 1,3-propanediol or 1,4-anhydro-2-deoxy-D-ribitol were synthesized and used as primers for the polymerase chain reaction (PCR). Since these residues cannot be replicated by a DNA polymerase, the resulting PCR products have protruding 5' ends. Primers were designed with three regions, a 3' region complementary to the desired template, a 5' region complementary to a preselected nucleotide sequence, and a nonreplicable element interposed between these two containing 1-3 of the nonbase residues. The primers were used in a PCR and the products hybridized without denaturation to a solid support containing an immobilized preselected nucleotide sequence. Studies are reported showing the effects of the nonreplicable elements in primer extension reactions and the application to the capture of PCR products.

Base Sequence↗
PubMed1992

Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents.

Amino Acid Sequence↗
PubMed1992

Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation.

The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.

Base Sequence↗
PubMed1992

Recognition of foldback DNA by the human DNA (cytosine-5-)-methyltransferase.

In order to specify the recognition requirements of the human DNA (cytosine-5-)-methyltransferase, two isomeric 48mers were synthesized so as to link a long block of DNA with a shorter complementary block of DNA through a tether consisting of five thymidine residues. These isomeric foldback molecules, differing only in the location of the 5-methyldeoxycytosine, were shown to be unimolecular, to contain a region of duplex DNA, and to contain a region of single-stranded DNA. When used as substrates for the DNA methyltransferase, only one of the isomers was methylated. A comparison of the structures of the two isomers allows us to begin to define the potential sites of interaction between the enzyme and the three nucleotides forming a structural motif consisting of 5-methyldeoxycytosine, its base-paired deoxyguanosine, and a deoxycytosine 5' to the paired deoxyguanosine.

Base Sequence↗
PubMed1992

Detection of specific alleles by using allele-specific primer extension followed by capture on solid support.

This article describes a method for determining whether a particular nucleic acid sequence is present in a sample and for discriminating between any two nucleic acid sequences if such sequences differ only by a single nucleotide. The method entails extension of a novel two-component primer on templates that may or may not include a target nucleic acid sequence. The 3' portion of the primer is complementary to a portion of the template adjacent to the target sequence (for example, the polymorphic nucleotide). The 5' portion of the primer is complementary to a different preselected nucleic acid sequence. Extension of the 3' portion of the primer with a labeled deoxynucleoside triphosphate yields a labeled extension product, but only if the template includes the target sequence. The presence of such a labeled primer-extension product is detected by hybridization of the 5' portion to the preselected sequence. The preselected sequence is immobilized on a solid support. The method has been applied to genotyping individuals for the two-allele polymorphism of the human tyrosinase gene.

Alleles↗
PubMed1991

Recognition of unusual DNA structures by human DNA (cytosine-5)methyltransferase.

The symmetry of the responses of the human DNA (cytosine-5)methyltransferase to alternative placements of 5-methylcytosine in model oligodeoxynucleotide duplexes containing unusual structures has been examined. The results of these experiments more clearly define the DNA recognition specificity of the enzyme. A simple three-nucleotide recognition motif within the CG dinucleotide pair can be identified in each enzymatically methylated duplex. The data can be summarized by numbering the four nucleotides in the dinucleotide pair thus: 1 4/2 3. With reference to this numbering scheme, position 1 can be occupied by cytosine or 5-methylcytosine; position 2 can be occupied by guanosine or inosine; position 3, the site of enzymatic methylation, can be occupied only by cytosine; and position 4 can be occupied by guanosine, inosine, O6-methylguanosine, cytosine, adenosine, an abasic site, or the 3' hydroxyl group at the end of a gapped molecule. Replacing the guanosine normally found at position 4 with any of the moieties introduces unusual (non-Watson-Crick) pairing at position 3 and generally enhances methylation of the cytosine at that site. The exceptional facility of the enzyme in actively methylating unusual DNA structures suggests that the evolution of the DNA methyltransferase, and perhaps DNA methylation itself, may be linked to the biological occurrence of unusual DNA structures.

5-Methylcytosine↗
PubMed1990

The abasic site as a challenge to DNA polymerase. A nuclear magnetic resonance study of G, C and T opposite a model abasic site.

An abasic site in DNA creates a strong block to DNA polymerase and is a mutagenic base lesion. In this study, we present structural and dynamic properties of duplex oligodeoxynucleotides containing G, C and T opposite a model abasic site studied by one and two-dimensional nuclear magnetic resonance spectroscopy. We have demonstrated that A opposite the abasic site was positioned within the helix as if paired with T, and that the A residue melted co-operatively with the surrounding helix. We report here that G opposite the abasic site is also observed to be predominantly intrahelical in a normal anti conformation at low temperature. With increasing temperature, the mobility of the G residue increases rapidly and apparently is in a "melted state" well before denaturation of the helix. At low temperature, two species are found for T opposite the abasic site; one, intrahelical, one extrahelical. These species are in slow exchange with one another on a proton nuclear magnetic resonance time-scale. The two species then move into fast exchange with increasing temperature and the proportion of the extra-helical form increases. When C is positioned opposite the abasic site, both the C residue and the abasic sugar are extrahelical, the helix collapses, and the adjacent G.C base-pairs stack over one another. On the basis of these observations, we propose a model that explains why the abasic site acts to block DNA replication. Further, we suggest an explanation for the observed polymerase preference for base selection at abasic sites.

Base Sequence↗
PubMed1990

Identification of a ligand-binding region of the human insulin receptor encoded by the second exon of the gene.

Structure-function studies of the insulin molecule indicate that an insulin B chain domain comprising residues 22-26 is involved both in binding to the insulin receptor (INSR) and in insulin dimer formation, suggesting that this domain might also interact with a structure resembling the insulin dimer interface in the INSR. Expression of a mutant INSR cDNA with a deletion of the region corresponding to exon 2 of the INSR gene produces a protein devoid of insulin-binding activity, although the mutant protein is processed appropriately to alpha- and beta-subunits, suggesting that the insulin-binding domain is encoded at least in part by exon 2. Within this region of the INSR molecule, the sequence 83-103 fulfills the structural criteria for a dimer interface. Studies of mutant INSRs with substitutions for phenylalanine 88 or 89 show that the presence of phenylalanine at position 89 is essential for full binding affinity.

Amino Acid Sequence↗
PubMed1989

Characterization of the high pH wobble structure of the 2-aminopurine.cytosine mismatch by N-15 NMR spectroscopy.

Transition mutations induced by the base analogue 2-aminopurine arise via the formation of AP.C base pairs during DNA replication. We report here the results of N-15 NMR studies on a duplex oligonucleotide containing N-15 enriched AP and C residues. At high pH (8.6) the AP.C base pair is predominantly wobble. This is the first report on use of a site specifically N-15 enriched oligonucleotide as a probe of aberrant base pairing in DNA.

2-Aminopurine↗
PubMed1988

Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA.

We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus. A DNA polymerase activity has been purified from E. coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid. Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E. coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-). The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis. Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III). In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit). Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III. Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein. Pol X has a molecular mass of 84 kDa. Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E. coli.

Base Sequence↗