Biomedical subjects

E B Kmiec

Publications and source records attributed to E B Kmiec.

At least 55 records · Page 3Linked to original sources

PubMed1994

DNA strand exchange in the absence of homologous pairing.

The strand exchange reaction that is widely used for in vitro studies on recombination of DNA molecules is generally presumed to result from a preceding homologous pairing step. With the use of a single-stranded circular DNA and a short homologous linear duplex fragment as substrates in a model strand exchange reaction, it was found that modest concentrations of polyethylene glycol or salt promote formation of heteroduplex molecules and strand exchange. When the duplex fragment was partially resected with an exonuclease exposing a short single-stranded stretch on the ends, the reaction was promoted by the addition of commercial bovine serum albumin. The transcription factor TFIIIA promoted strand exchange when the DNA substrates contained the cognate DNA binding sequence recognized by the protein. These observations suggest that detection of strand exchange in vitro does not necessarily imply a preceding homologous pairing step.

Animals↗

PubMed1994

ATP-independent DNA renaturation catalyzed by a protein from Ustilago maydis.

A protein that catalyzes the renaturation of complementary strands of DNA has been purified from mitotic cells of the lower eukaryote Ustilago maydis. The most highly purified fraction contains a polypeptide with a molecular mass of 20 kDa as determined by SDS/PAGE and glycerol gradient sedimentation. DNA reannealing is enhanced by the presence of a divalent cation but does not require ATP nor any other nucleotide triphosphate. Reassociation proceeds with fast kinetics as more than 60% of the DNA is reannealed within 4 min at a 30:1 nucleotide/protein monomer ratio, results which suggest that the protein acts in a stoichiometric fashion. Amino acid analysis revealed that the protein contained an elevated level of basic residues and low levels of tryptophan and tyrosine. The protein binds to an oligonucleotide of ten residues but not to one having only five. As judged by agarose gel assays, the protein does not catalyze strand-transfer reactions but does promote the annealing of a 58-residue polynucleotide onto single-stranded circles and gapped linear duplexes. These latter reactions are dependent on the presence of DNA sequence similarity between the pairing partners.

Adenosine Triphosphate↗

PubMed1994

ATP-dependent DNA renaturation and DNA-dependent ATPase reactions catalyzed by the Ustilago maydis homologous pairing protein.

Purification of the ATP-dependent homologous pairing activity from Ustilago maydis yields a protein preparation that is enriched for a 70-kDa polypeptide as determined by SDS-gel electrophoresis. The protein responsible for the ATP-dependent pairing activity, using renaturation of complementary single strands of DNA as an assay, has a Stokes radius of 3.6 nm and a sedimentation coefficient of 4.3 S consistent with the interpretation that the activity arises from a monomeric globular protein of 70 kDa. Including heparin-agarose and FPLC gel filtration chromatography steps in the previously published protocol improves the purification of the protein. ATP and Mg2+ are necessary cofactors for optimal DNA renaturation activity. ADP inhibits the reaction. Analysis of the ATP-dependent renaturation kinetics indicates the reaction proceeds through a first-order mechanism. The protein has an associated DNA-dependent ATPase as indicated by co-chromatography with the purified ATP-dependent renaturation activity through an FPLC gel-filtration column. Single-stranded DNA and Mg2+ are required for optimal ATP hydrolytic activity, although a number of other polynucleotides and divalent cations can substitute to varying degrees. Hydrolysis of ATP is activated in a sigmoidal manner with increasing amounts of the protein. At ATP concentrations below 0.1 mM the ATPase activity exhibits positive cooperativity as indicated from the Hill coefficient of 1.8 determined by steady-state kinetic analysis of the reaction. ADP and adenosine 5'-[beta,gamma-imido]triphosphate are inhibitors of the ATPase activity although they appear to exert their inhibitory effects through different modes. These results are interpreted as evidence for protein-protein interactions.

Adenosine Triphosphatases↗

PubMed1994

The REC2 gene encodes the homologous pairing protein of Ustilago maydis.

Amino acid sequence analysis has established that the homologous pairing protein of Ustilago maydis, known previously in the literature as rec1, is encoded by REC2, a gene essential for recombinational repair and meiosis with regional homology to Escherichia coli RecA. The 70-kDa rec1 protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously during sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product, the REC2 gene was overexpressed from a vector that fused a hexahistidine leader sequence onto the amino terminus, enabling isolation of the REC2 protein on an immobilized metal affinity column. The purified protein exhibits ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from cell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds between identical DNA strands. A size threshold of about 50 bp was found to govern pairing between linear duplex molecules and homologous single-stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the duplex was composed of RNA. Linear duplex molecules with hairpin caps also formed joint molecules when as few as three RNA residues were present.

Amino Acid Sequence↗

PubMed1994

Transcription activates RecA-promoted homologous pairing of nucleosomal DNA.

RecA protein catalyzes the homologous pairing of a single-stranded circular DNA and a linear duplex DNA molecule. When the duplex is packaged into chromatin, formation of homologously paired complexes is blocked. We have established a system for studying the RecA-promoted reaction by using a duplex fragment containing a single-phased nucleosome. Under these conditions there is no reaction leading to formation of joint molecule complexes. However, transcription on the chromatin template activates the formation of complexes. Reaction is dependent on RNA synthesis and DNA sequence homology and proceeds regardless of the direction of transcription.

Cell-Free System↗

PubMed1994

A role for RNA synthesis in homologous pairing events.

The relationship between RNA synthesis and homologous pairing in vitro, catalyzed by RecA protein, was examined by using an established strand transfer assay system. When a short DNA duplex is mixed with single-stranded circles, RecA protein promotes the transfer of the minus strand of the duplex onto the complementary region of the plus-strand circle, with the displacement of the plus strand of the duplex. However, if minus-strand RNA is synthesized from the duplex pairing partner, joint molecules containing the RNA transcript, the plus strand of the DNA duplex, and the plus-strand circle are also observed to form. This reaction, which is dependent on RNA polymerase, sequence homology, and RecA protein, produces a joint molecule that can be dissolved by treatment with RNase H but not RNase A. Under these reaction conditions, product molecules form even when the length of shared homology between duplex and circle is reduced to 15 bp.

DNA, Circular↗

PubMed1993

Characterization of Ustilago maydis DNA binding protein one (UBP1).

The DNA binding properties of a protein from the lower eukaryote Ustilago maydis have been characterized. Using both filter binding and gel retention assays, we demonstrate that this protein, termed UBP1 (Ustilago binding protein one), binds preferentially to DNA molecules lacking chain interruptions. The introduction of DNA breaks by a restriction enzyme or a purified nuclease, from Ustilago maydis, causes the dissociation of protein-DNA complexes. UBP1 stimulates the relaxation of negatively supercoiled DNA, mediated by Ustilago type I topoisomerase, through a mechanism most likely involving the association of UBP1 with the DNA rather than with the topoisomerase. The prebinding of UBP1 to DNA templates, subsequently assembled into minichromosomes, results in the development of a disorganized nucleosomal array. Possible roles for UBP1 in processes that involve changes in DNA topology, such as chromatin assembly, are discussed.

DNA↗

PubMed1993

DNA relaxation mediated by Ustilago maydis type I topoisomerase; modulation by chromatin associated proteins.

Ustilago maydis topoisomerase I relaxes superhelical DNA in the absence of any co-factors. The reaction reaches a defined end-point proportional to the amount of enzyme added and an analysis of the reaction by Hill plot transformation indicates that at least two molecules of topoisomerase must interact with the DNA to catalyze relaxation. The addition of purified Ustilago histone H1 reduces the stoichiometric amount of topoisomerase I required by 50%. H1 histone may function to enhance DNA relaxation through a cooperative mechanism. The purified HMG-like protein from Ustilago also enhances DNA relaxation mediated by the topoisomerase. Whereas H1 stimulates topo I-mediated DNA relaxation through a processive mode, the HMG-like protein enhances through a distributive mechanism. Taken together, these results demonstrate that the interaction of chromosomal proteins with topoisomerase can influence DNA topology, and mechanisms are proposed to explain this enhancement.

DNA Topoisomerases, Type I↗

PubMed1993

The glucocorticoid receptor precludes the binding of a transcriptional repressor protein to the long terminal repeat of the mouse mammary tumor virus.

The long terminal repeat (LTR) of the mouse mammary tumor virus was used as a template to examine the dual binding parameters of the glucocorticoid-receptor (GR) and a repressor protein termed Inhibitory Factor 1 (IF1). The receptor binds specifically to the glucocorticoid response element and precludes the binding of IF1 to its juxtaposed binding site within the LTR. When the two DNA targets are separated by the insertion of an additional 52 base pairs, coincident binding of both proteins is observed. Gel retention assays reveal three distinct nucleoprotein complexes. The first complex consists of the receptor and the LTR, the second is comprised of IF1 and DNA and the third is a multiprotein-DNA complex consisting of the GR, IF1 and DNA, migrating at a higher molecular weight position. The inhibition of IF1 binding by the presence of prebound GR leads to the repression of transcription of juxtaposed genes. The GR may act to block access of a sequence, used by the cell to titrate repressor proteins and facilitate the onset of gene expression.

Animals↗

PubMed1993

Positive regulation of tRNA gene expression by the mouse mammary tumor virus-long terminal repeat in vitro.

The mouse mammary tumor virus long terminal repeat (MMTV-LTR) participates in the control of gene expression by providing a series of important DNA binding sites at which trans-acting factors interact. Among these factors are the steroid receptor, nuclear factor I (NFI) and the TATA box factor (TFIID). The binding of these proteins facilitates the assembly of a transcriptionally competent complex, that includes RNA polymerase II, and activates the expression of juxtaposed genes in cis. A particular DNA sequence, distinct from previously identified regulatory elements, was found in the present study to activate gene expression in trans. The sequence is located between nucleotides +3 and +43 near the 3' terminus of the LTR. This sequence binds a protein that may actively repress the expression of genes that are not located immediately in cis. This protein was purified by ion exchange chromatography and has an approximate molecular weight of 31,000 daltons, as judged by SDS-PAGE. Gel retardation experiments reveal that progressively larger protein--DNA complexes are formed when the amount of this factor is increased relative to the DNA binding site. Furthermore, this protein was found to preferentially aggregate DNA molecules containing the LTR sequence between bases +3 and +43. These results reveal the existence of a unique modulatory role for the LTR in regulating gene expression in trans.

Animals↗

PubMed1993

In vitro transcription of the c-myc first exon may be influenced by the extent of chromatin assembly.

The first exon of the human c-myc gene can be transcribed by either RNA polymerase II or RNA polymerase III. The molecular factors contributing to polymerase selection are not yet completely defined. We have examined the role of chromatin structure in regulating transcription by RNA polymerase III. Using as competitor a pol III gene in both a cis and trans arrangement, we demonstrate that c-myc gene expression is facilitated from templates containing a minimal number of fully assembled nucleosomes. The removal of excess histones by DNA titration leads to an elevated level of c-myc expression. These results suggest that either the c-myc expression is inhibited when the template is fully packaged into chromatin or that the affinity of RNA polymerase for the regulatory elements of this exon is such that a template, devoid of histones, is required for transcriptional initiation.

Animals↗

PubMed1993

Purification and properties of a cruciform DNA binding protein from Ustilago maydis.

A DNA binding protein with an M(r) of 11,000 was purified from Ustilago maydis. Its solubility in acid, amino acid composition, and mobility during gel electrophoresis are reminiscent of properties observed for the high mobility group nonhistone chromosomal proteins. The protein recognizes cruciform DNA made from oligonucleotides and also binds preferentially to a plasmid containing an extruded cruciform.

Base Sequence↗

PubMed1993

TFIIIA and DNA supercoiling: toward resolving a controversy.

DNA supercoiling in eukaryotes is mediated by the process of chromatin assembly. This process couples the binding and wrapping of DNA around nucleosomal core particles and the enzymatic activity of DNA topoisomerase(s). Kmiec and Worcel (1985) reported that the binding of an RNA polymerase III transcription factor (TFIIIA) could accelerate DNA supercoiling catalysed by a Xenopus laevis cell-free extract. Although the reaction was repeatedly reproduced in the parent laboratory by numerous workers, another laboratory, interested in the molecular biology of TFIIIA, failed to reproduce TFIIIA-mediated supercoiling or gyration. In this review, an attempt is made to transcend personal beliefs and describe the experimental approaches used to clarify this issue. The original variability lay in the amount of endogenous RNA within the cell free extract and the concentrations of MgCl2 used to conduct the experiments.

Adenosine Triphosphate↗

PubMed1992

In vitro chromatin assembly promoted by the Xenopus laevis S-150 cell-free extract is enhanced by treatment with RNase A.

Cell-free extracts employed as chromatin assembly systems contain a myriad of proteins, polyanions and nucleic acids. The roles of ATP, MgCl2 and other cofactors in the catalysis of nucleosome formation by the Xenopus laevis oocyte S-150 have yet to be established unequivocally. In this study we examine the influence of RNA in the assembly process. Under reaction conditions that inhibit nucleosome formation (+ EDTA), pretreatment of the extract with RNase A revives the chromatin assembly machinery while the rate of DNA supercoiling is stimulated significantly. Addition of purified RNA blocks DNA supercoiling. Taken together, these data suggest that the parameters surrounding in vitro chromatin assembly are variable and subject to modulation by endogenous factors.

Adenosine Triphosphate↗

PubMed1992

In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells.

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl2, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.

Animals↗

PubMed1991

An analysis of transcription factor TFIIIA-mediated DNA supercoiling.

We have analyzed transcription factor-mediated DNA supercoiling catalyzed by the Xenopus oocyte extract (S-150). Under conditions that inhibit endogenous supercoiling activity (2 mM EDTA), the 5S RNA specific transcription factor, TFIIIA, promotes a negative change in DNA linking number. The SV40 binding protein, T antigen, appears not to promote DNA supercoiling under these conditions. A nucleosomal ladder can be seen after DNase I digestions only if the DNA template is pre-bound by TFIIIA prior to the addition of the S-150 extract. These studies suggest that TFIIIA may stimulate DNA supercoiling by enhancing the development of protein-DNA interactions via a mechanism that may include nucleosome assembly.

Animals↗

PubMed1990

Transcription factor TFIIIA stimulates DNA supercoiling promoted by a fractionated cell-free extract from Xenopus laevis.

An activity that can introduce negative supercoils into relaxed covalently closed DNA molecules has been isolated from a Xenopus laevis cell-free extract (S-150) and purified over 200-fold. The exogenous addition of ATP, other ribonucleotides and deoxyribonucleotides, as well as nonhydrolyzable analogs, stimulate DNA supercoiling which may occur by a pathway involving multiple protein components. DNA supercoiling occurs in topological single steps and is inhibited by camptothecin and berenil, but not novobiocin or VM-26, suggesting a catalytic role for topoisomerase I in the reaction. Transcription factor TFIIIA stimulates DNA supercoiling catalyzed by the isolated fraction at low factor to DNA ratios. Taken together, these results suggest that a isolated DNA supercoiling activity from the X. laevis S-150 cell-free extract can be stimulated by transcription factor TFIIIA.

Adenosine Triphosphate↗

PubMed1990

Reaction parameters of TFIIIA-induced supercoiling catalyzed by a Xenopus laevis cell-free extract.

In addition to its fundamental role of nucleating the formation of stable transcription complexes, the Xenopus laevis 5S RNA specific transcription factor, TFIIIA, promotes a variety of DNA-associated metabolic reactions. We report that TFIIIA can induce a DNA supercoiling catalyzed by the Xenopus laevis S-150 cell-free extract on plasmids containing a single copy of the Xenopus 5S RNA gene (somatic-type). Stimulated supercoiling occurs in the presence of high concentrations of ATP (4 mM) and at a factor to DNA ratio of 1 through a mechanism most likely involving type I topoisomerase. The highest level of stimulated supercoiling occurs when TFIIIA is incubated with DNA prior to the addition of the S-150 extract. Taken together, the experiments outlined in this report establish a reliable and seminal system in which TFIIIA-induced DNA supercoiling can be observed reproducibly.

Adenosine Triphosphate↗