Sequence-specific Single-stranded DNA Research Articles

Association of HIV-1 Tat with the cellular protein, Puralpha, is mediated by RNA.

The interaction between two regulatory proteins plays a crucial role in the control of several biological events, including gene transcription. In this report, we demonstrate that the interaction between the cellular sequence-specific single-stranded DNA binding protein Puralpha and the HIV type 1 (HIV-1) Tat protein is mediated by specific ribonucleic acids. The region of Tat that is important for its interaction with Puralpha includes the region demonstrated to bind Tat's viral RNA target, TAR. A 10-nucleotide GC-rich consensus sequence identified in RNAs associated with Puralpha derived from human U-87MG cells plays an important role in the Puralpha:Tat interaction as examined by an in vitro reconstitution assay. Furthermore, expression of the Puralpha-associated RNA in these cells enhances transcriptional activation of the HIV-1 promoter by Tat and Puralpha.

The Cys4 zinc finger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition.

Bacteriophage T7 DNA primase recognizes 5'-GTC-3' in single-stranded DNA. The primase contains a single Cys4 zinc-binding motif that is essential for recognition. Biochemical and mutagenic analyses suggest that the Cys4 motif contacts cytosine of 5'-GTC-3' and may also contribute to thymine recognition. Residues His33 and Asp31 are critical for these interactions. Biochemical analysis also reveals that T7 primase selectively binds CTP in the absence of DNA. We propose that bound CTP selects the remaining base G, of 5'-GTC-3', by base pairing. Our deduced mechanism for recognition of ssDNA by Cys4 motifs bears little resemblance to the recognition of trinucleotides of double-stranded DNA by Cys2His2 zinc fingers.

Selection of novel, specific single-stranded DNA sequences by Flp, a duplex-specific DNA binding protein.

Flp is a member of the integrase family of site-specific recombinases. Flp is known to be a double-stranded (ds)DNA binding protein that binds sequence specifically to the 13 bp binding elements in the FRT site (Flprecognitiontarget). We subjected a random pool of oligonucleotides to the in vitro binding site selection method and have unexpectedly recovered a series of single-stranded oligonucleotides to which Flp binds with high affinity. These single-stranded oligonucleotides differ in sequence from the duplex FRT site. The minimal length of the oligonucleotides which is active is 29 nt. This single strand-specific DNA binding activity is located in the same C-terminal 32 kDa domain of Flp in which the site-specific dsDNA binding activity resides. Competition studies suggest that the apparent affinity of Flp for single-stranded oligonucleotide is somewhat less than for a complete duplex FRT site but greater than for a single duplex 13 bp binding element. We have also shown that Cre, another member of the integrase family of site-specific recombinases, also exhibits single-stranded DNA binding similar to that of Flp.

Activation of the JC virus Tat-responsive transcriptional control element by association of the Tat protein of human immunodeficiency virus 1 with cellular protein Pur alpha.

JC virus is activated to replicate in glial cells of many AIDS patients with neurological disorders. In human glial cells, the human immunodeficiency virus 1 (HIV-1) Tat protein activates the major late promoter of JC virus through a Tat-responsive DNA element, termed upTAR, which is a recognition site for cellular Pur alpha, a sequence-specific single-stranded DNA binding protein implicated in cell cycle control of DNA replication and transcription. Tat interacts with two leucine-rich repeats in Pur alpha to form a complex that can be immunoprecipitated from cell extracts. Tat enhances the ability of purified glutathione S-transferase-Pur alpha (GST-Pur alpha) to bind the upTAR element. Tat acts synergistically with Pur alpha, in a cell-cycle-dependent manner, to activate transcription at an upTAR element placed upstream of a heterologous promoter. Since Pur alpha is ubiquitously expressed in human cells and since PUR elements are located near many promoters and origins of replication, the Tat-Pur alpha interaction may be implicated in effects of HIV-1 throughout the full range of HIV-1-infected cells.

Repression of Transcriptional Enhancer Factor-1 and Activator Protein-1-dependent Enhancer Activity by Vascular Actin Single-stranded DNA Binding Factor 2

Transcriptional repression of the murine vascular smooth muscle alpha-actin gene in fibroblasts results from the interaction of two sequence-specific single-stranded DNA binding activities (VACssBF1 and VACssBF2) with opposite strands of an essential transcriptional enhancer factor-1 (TEF-1) element (Sun, S., Stoflet, E. S., Cogan, J. G., Strauch, A. R., and Getz, M. J. (1995) Mol. Cell. Biol. 15, 2429-2436). Here, we identify a sequence element located within a protein-coding exon of the gene that bears structural similarity with the TEF-1 enhancer. This includes a 30-base pair region of purine-pyrimidine asymmetry encompassing a perfect 6-base pair GGAATG TEF-1 recognition motif. Unlike the enhancer, however, the exon sequence exhibits no TEF-1 binding activity nor does the pyrimidine-rich strand bind VACssBF1. However, VACssBF2 interacts equally well with the purine-rich strand of both the enhancer and the exon sequence. To test the ability of VACssBF2 to independently repress transcription, the exon sequence was placed upstream of a deletionally activated promoter containing an intact TEF-1 binding site. The exon sequence repressed promoter activity, whereas a mutant deficient in VACssBF2 binding did not. Moreover, VACssBF2 similarly repressed activator protein-1-dependent transcription of a heterologous tissue factor promoter. These results suggest that VACssBF2 possesses an intrinsic ability to disrupt enhancer function independently of the enhancer-binding proteins involved.

P-123 - Functional characterization of ssCRE-BP, a sequence-specific single-stranded DNA binding protein

P-123 - Functional characterization of ssCRE-BP, a sequence-specific single-stranded DNA binding protein

Identification of a Sequence-specific Single-stranded DNA Binding Protein That Suppresses Transcription of the Mouse Myelin Basic Protein Gene

The myelin basic protein (MBP) gene is expressed only in oligodendrocytes and Schwann cells, and expression follows a tightly regulated developmental time course. Cell type- and developmental stage-specific expression of the MBP gene appears to be regulated by a series of cis-acting elements located upstream of the transcription start site. The proximal element of the MBP regulatory region (MB1), located between nucleotides -14 and -50, is one of several elements participating in the programmed expression of MBP. In this report, we describe the molecular cloning and characterization of myelin gene expression factor-2 (Myef-2), a protein isolated from mouse brain that binds specifically to single-stranded DNA derived from the MB1 element and represses transcription of the MBP gene in transient transfection assay. Myef-2 mRNA is developmentally regulated in mouse brain; its peak expression occurs at postnatal day 7, prior to the onset of MBP expression. The developmental pattern of Myef-2 mRNA expression coincides with that previously described for SCIP, a POU domain transcription factor that also represses myelin basic protein expression. The myef-2 gene maps to mouse chromosome 2. The relevance of these findings for regulation of MBP gene expression and oligodendrocyte differentiation is discussed.

The single-stranded DNA aptamer-binding site of human thrombin.

A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.