Polypurine-polypyrimidine Tract Research Articles

Mechanism of Strand-Specific Smooth Muscle α-Actin Enhancer Interaction by Purine-Rich Element Binding Protein B (Purβ)

Expression of the smooth muscle alpha-actin gene in growth-activated vascular smooth muscle cells and stromal fibroblasts is negatively regulated by members of the Pur family of single-stranded DNA/RNA-binding proteins. In particular, Puralpha and Purbeta are postulated to repress transcription by forming helix-destabilizing complexes with the sense strand of an asymmetric polypurine-polypyrimidine tract containing a canonical MCAT enhancer motif in the 5' region of the gene. Herein, we establish the mechanism of Purbeta binding to the purine-rich strand of the enhancer using quantitative methods and purified components. Initial evaluation of DNA-binding specificity and equilibrium stoichiometry via colorimetric-, autoradiographic-, and fluorescence-based assays suggested that Purbeta interacts with two distinct G/A-rich sites within the nominal single-stranded enhancer element to form a high-affinity 2:1 protein:DNA complex. Statistical mechanical analyses of band shift titrations of the nominal element in conjunction with DNase I footprint titrations of the extended smooth muscle alpha-actin 5'-flanking region demonstrated that assembly of the nucleoprotein complex likely occurs in a sequential, cooperative, and monomer-dependent fashion. Resolution of the microscopic energetics of the system indicated that monomer association with two nonidentical sites flanking the core MCAT motif accounts for the majority of the intrinsic binding affinity of Purbeta with intersite cooperativity contributing an approximately 12-fold increase to the stability of the nucleoprotein complex. These findings offer new insights into the mechanism, energetics, and sequence determinants of Purbeta repressor binding to a biologically relevant, contractile phenotype-regulating cis-element while also revealing the thermodynamic confines of putative Purbeta-mediated effects on DNA structure.

Using nucleases to stimulate homologous recombination.

In essentially all organisms, double-strand breaks in chromosomal DNA stimulate repair by multiple mechanisms, including homologous recombination. It is possible to use site-specific reagents to produce a break or other recombinagenic damage at a unique site, which makes possible detailed analysis of the repair products. In addition, targeted mutagenesis and gene replacement are stimulated in the immediate vicinity of the break site. To utilize meganucleases with long recognition sequences, it is necessary to introduce the corresponding sequence prior to directed cleavage. The same is typically true of triplex-forming oligonucleotides that target polypurine-polypyrimidine tracts. Zinc-finger nucleases have the potential of being targetable to arbitrarily selected sites, owing to the flexibility of zinc finger recognition of DNA.

Altered Sensitivity to Single-strand-specific Reagents Associated with the Genomic Vascular Smooth Muscle α-Actin Promoter during Myofibroblast Differentiation

Stimulation of quiescent AKR-2B mouse fibroblasts with transforming growth factor beta1 results in uniform conversion to a myofibroblast-like phenotype as judged by a rapid accumulation of smooth muscle alpha-actin mRNA and protein. Because transcriptional regulation of the smooth muscle alpha-actin gene in these cells might be mediated by single-stranded DNA-binding proteins, we have examined the sensitivity of genomic DNA to chemical reagents with specificity for unpaired bases in a region of the promoter previously implicated in Puralpha, Purbeta, and MSY1 binding in vitro (Kelm, R. J., Jr., Cogan, J. G., Elder, P. K., Strauch, A. R., and Getz, M. J. (1999) J. Biol. Chem. 274, 14238-14245). Our data reveal specific differences between purified DNA treated in vitro and nucleoprotein complexes treated in living cells. Although some differences were observed in quiescent cells, treatment with transforming growth factor beta1 resulted in the development of additional sensitivity within 1 h. This enhancement was most pronounced in bases immediately upstream of an MCAT enhancer element-containing polypurine-polypyrimidine tract. A TATA-proximal element of similar base distribution showed no such hyperreactivities. These results suggest that activation of the endogenous smooth muscle alpha-actin gene during myofibroblast conversion is accompanied by specific structural changes in the promoter that are consistent with a decline in single-stranded DNA repressor protein binding.

A triplex-mediated knot between separated polypurine-polypyrimidine tracts in circular DNA blocks transcription by Escherichia coli RNA polymerase.

Polypurine-polypyrimidine tracts are overrepresented in eukaryotes and many have the potential to form triplex DNA. Transmolecular triplexes form between separated but complementary polypurine-polypyrimidine tracts in duplex DNA. Transmolecular triplexes (T-loops) were studied previously using a circular plasmid containing a pair of separated polypurine-polypyrimidine tracts designed to able to form a triplex with each other. T-Loops formed when the nicked plasmid was incubated at low pH in the presence of spermine. When the pH was raised to 8, the T-loops were constrained by a hydrogen-bonded knot composed of multistranded and single-stranded regions. The present experiments used T-loops as a model system to investigate the influence of transmolecular triplex formation on transcription. T-Loops and control open circular, linear, and supercoiled plasmid forms were isolated from bands on agarose gels. Transcription assays were carried out with the isolated plasmid forms and Escherichia coli RNA polymerase holoenzyme and the core enzyme, which lacked sigma70. Transcription was significantly inhibited in T-loop forms compared with control plasmid forms. There was no evidence that the single-stranded regions of T-loops facilitated nonspecific initiation of transcription. Instead, the multistranded component of the hydrogen-bonded knot at the root of the T-loop structure inhibited transcription.

Human amiloride-sensitive epithelial Na+ channel γ subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation

Human amiloride-sensitive epithelial Na+ channel γ subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation

Human amiloride-sensitive epithelial Na+ channel γ subunit promoter: functional analysis and identification of a polypurine-polypyrimidine tract with the potential for triplex DNA formation

The mRNA for the epithelial Na(+) channel gamma subunit (gammaENaC) is regulated developmentally in the lung, colon and distal nephron and in response to Na(+) deprivation and systemic corticosteroids in the distal colon. Because such regulation is likely to be at the level of gene transcription, we examined the function of the promoter and other 5' flanking elements of the human gammaENaC gene. The proximal 5' flanking region contains two GC boxes but does not contain a TATA box. A 450 bp human gammaENaC fragment (-459 to +40) directed the expression of luciferase in H441 cells and primer extension analysis in transfected cells confirmed the correct initiation of human gammaENaC-luciferase chimaeric transcripts. By deletional analysis, GC boxes at -21 and -52 were found to be critical for this promoter activity. To begin to identify transcription factors that bind to the core promoter, a double-stranded oligonucleotide that corresponded to this region was synthesized and tested in a gel mobility-shift assay. Incubation of this radiolabelled oligonucleotide with nuclear extracts from H441 and FRTL5 cells resulted in the formation of four specific and distinct DNA-protein complexes. On the basis of antibody 'supershift' assays, one of these factors corresponds to Sp1, whereas the other three correspond to Sp3. Further upstream, an approx. 300 nt (-1143 to -839) polypurine-polypyrimidine tract (PPy tract) containing internal mirror repeats was identified. When contained in a supercoiled plasmid, the approx. 1200 nt 5' flanking region was sensitive to S1 endonuclease, which was consistent with the formation of an intramolecular triplex DNA ('H-DNA') structure with an unpaired single strand. High-resolution mapping with S1 endonuclease and sequencing of S1-generated clones confirmed that all S1-sensitive sites were within the PPy tract. Finally, a negative regulatory element was identified between -1525 and -1296 that functioned in lung, colon and collecting duct cell lines.

Molecular Interactions between Single-stranded DNA-binding Proteins Associated with an Essential MCAT Element in the Mouse Smooth Muscle α-Actin Promoter

Transcriptional activity of the mouse vascular smooth muscle alpha-actin gene in fibroblasts is regulated, in part, by a 30-base pair asymmetric polypurine-polypyrimidine tract containing an essential MCAT enhancer motif. The double-stranded form of this sequence serves as a binding site for a transcription enhancer factor 1-related protein while the separated single strands interact with two distinct DNA binding activities termed VACssBF1 and 2 (Cogan, J. G., Sun, S., Stoflet, E. S., Schmidt, L. J., Getz, M. J., and Strauch, A. R. (1995) J. Biol. Chem. 270, 11310-11321; Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2936). VACssBF2 has been recently cloned and shown to consist of two closely related proteins, Puralpha and Purbeta (Kelm, R. J., Elder, P. K., Strauch, A. R., and Getz, M. J. (1997) J. Biol. Chem. 272, 26727-26733). In this study, we demonstrate that Puralpha and Purbeta interact with each other via highly specific protein-protein interactions and bind to the purine-rich strand of the MCAT enhancer in the form of both homo- and heteromeric complexes. Moreover, both Pur proteins interact with MSY1, a VACssBF1-like protein cloned by virtue of its affinity for the pyrimidine-rich strand of the enhancer. Interactions between Puralpha, Purbeta, and MSY1 do not require the participation of DNA. Combinatorial interactions between these three single-stranded DNA-binding proteins may be important in regulating activity of the smooth muscle alpha-actin MCAT enhancer in fibroblasts.

Large deletions induced in the white gene of Drosophila melanogaster by the antitumoral drug cis-dichlorodiammineplatinum(II): influence of non-homologous recombination

We have studied two mutants carrying large deletions induced in the white gene of Drosophila by the antitumoral drug cisplatin. The breakpoints of the deletions were located by southern analysis and the sequences of the deletion junctions were determined. Two base-pair repeats are associated with the ends of these deletions; one of the repeats is preserved in the new junction after the deletion. DNA sequences such as A-T rich, alternating purine/pyrimidine tracts, polypurine-polypyrimidine tracts and topoisomerase I and II cleavage sites are found near the junctions. These results suggest that illegitimate recombinatinal processes are involved in the generation of cisplatin-induced large deletions.

DNA-stacking interactions Determine the Sequence Specificity of the Deoxyribonuclease Activity of 1,10-Phenanthroline-copper Ion

Bis(1,10-phenanthroline)-copper(I) ion (OP 2Cu +) binds reversibly to B-DNA and makes single-stranded cuts by oxidative attack on the deoxyribose moiety. The deoxyribonuclease activity is sequence-depen dent yet not nucleotide-specific at the cutting site. OP 2Cu +sequence specificity was analysed in terms of local variations of DNA stability. Kinetic constants of strand cleavage were measured at sequence positions on the two strands and converted into activation free energies of the cleavage reaction. DNA unwinding free energies were calculated from the base sequence using B-DNA stacking parameters for calculations. The two free-energy variations were statistically compared for a series of DNA restriction fragments bearing the binding sites of regulatory proteins and representing a total of 345 DNA base positions. This study shows that the mean activation free energy of strand cleavage at a pair of opposing sugars across the DNA minor groove varies like the unwinding free energy of the DNA sequence delimited by opposing sugars (3 to 4 bp). A statistical equality between the two free-energy variations is demonstrated when considering the sum of the two cleavage events at the opposing sugars. Systematic deviations between the two free-energy distributions were observed at specific sequences, including polypurine-polypyrimidine tracts (A n T m /A m T n , C n T m C p/G pA m G n ), alternating purine- pyrimidine tracts ((TA) n /(TA) n , (TG) n /(CA) n ) and at certain G + C-rich triplets (GGC, GCC and CGC). The physical significance of these observations is discussed and a model of OP 2Cu +binding and cleavage specificity based on the free-energy equality is proposed.

Negative regulation of the vascular smooth muscle alpha-actin gene in fibroblasts and myoblasts: disruption of enhancer function by sequence-specific single-stranded-DNA-binding proteins.

Transcriptional activation and repression of the vascular smooth muscle (VSM) alpha-actin gene in myoblasts and fibroblasts is mediated, in part, by positive and negative elements contained within an approximately 30-bp polypurine-polypyrimidine tract. This region contains binding sites for an essential transcription-activating protein, identified as transcriptional enhancer factor I (TEF-1), and two tissue-restrictive, sequence-specific, single-stranded-DNA-binding activities termed VACssBF1 and VACssBF2. TEF-1 has no detectable single-stranded-DNA-binding activity, while VACssBF1 and VACssBF2 have little, if any, affinity for double-stranded DNA. Site-specific mutagenesis experiments demonstrate that the determinants of VACssBF1 and VACssBF2 binding lie on opposite strands of the DNA helix and include the TEF-1 recognition sequence. Functional analysis of this region reveals that the CCAAT box-binding protein nuclear factor Y (NF-Y) can substitute for TEF-1 in activating VSM alpha-actin transcription but that the TEF-1-binding site is essential for the maintenance of full transcriptional repression. Importantly, replacement of the TEF-1-binding site with that for NF-Y diminishes the ability of VACssBF1 and VACssBF2 to bind to separated single strands. Additional activating mutations have been identified which lie outside of the TEF-1-binding site but which also impair single-stranded-DNA-binding activity. These data support a model in which VACssBF1 and VACssBF2 function as repressors of VSM alpha-actin transcription by stabilizing a local single-stranded-DNA conformation, thus precluding double-stranded-DNA binding by the essential transcriptional activator TEF-1.

Molecular and functional characterization of the promoter of ETS2, the human c-ets-2 gene.

The 5' end of the human c-ets-2 gene, ETS2, was cloned and characterized. The major transcription initiation start sites were identified, and the pertinent sequences surrounding the ETS2 promoter were determined. The promoter region of ETS2 does not possess typical "TATA" and "CAAT" elements. However, this promoter contains several repeat regions, as well as two consensus AP2 binding sites and three putative Sp1 sites. There is also a palindromic region similar to the serum response element of the c-fos gene, located 1400 base pairs (bp) upstream from the first major transcription initiation site. A G + C-rich sequence (GC element) with dyad symmetry can be seen in the ETS2 promoter, immediately following an unusually long (approximately 250-bp) polypurine-polypyrimidine tract. A series of deletion fragments from the putative promoter region were ligated in front of the bacterial chloramphenicol acetyltransferase gene and tested for activity following transfection into HeLa cells. The 5' boundary of the region needed for maximum promoter activity was found to be 159 bp upstream of the major initiation site. This region of 159 bp contains putative binding sites for transcription factors Sp1 and AP2 (one for each), the GC element, one small forward repeat, one inverted repeat, and half of the polypurine-pyrimidine tract. The promoter of ETS2 (within the polypyrimidine tract) serves to illustrate an alternative structure that may be present in genes with "TATA-less" promoters.