Naturally occurring poly(purine·pyrimidine) rich regions in the human genome are prone to adopt non-canonical DNA structures such as intramolecular triplexes (i.e. H-DNA). Such structure-forming sequences are abundant and can regulate the expression of several diseases-linked genes. In addition, the use of triplex-forming oligonucleotides (TFOs) to modulate gene structure and function has potential as an approach to targeted gene therapy. Previously, we found that endogenous H-DNA structures can induce DNA double-strand breaks and promote genomic rearrangements. Herein, we find that the DHX9 helicase co-immunoprecipitates with triplex DNA structures in mammalian cells, suggesting a role in the maintenance of genome stability. We tested this postulate by assessing the helicase activity of purified human DHX9 on various duplex and triplex DNA substrates in vitro. DHX9 displaced the third strand from a specific triplex DNA structure and catalyzed the unwinding with a 3′→5′ polarity with respect to the displaced third strand. Helicase activity required a 3′-single-stranded overhang on the third strand and was dependent on ATP hydrolysis. The reaction kinetics consisted of a pre-steady-state burst phase followed by a linear, steady-state pseudo-zero-order-reaction. In contrast, very little, if any helicase activity was detected on blunt triplexes, triplexes with 5′-overhangs, blunt duplexes, duplexes with overhangs, or forked duplex substrates. Thus, triplex structures containing a 3′-overhang represent preferred substrates for DHX9, where it removes the strand with Hoogsteen hydrogen-bonded bases. Our results suggest the involvement of DHX9 in maintaining genome integrity by unwinding mutagenic triplex DNA structures.
The ability of oligodeoxynucleotides to form specific triple helical structures with critical regulatory sequences in the human dihydrofolate reductase (DHFR) promoter was investigated. A battery of purine-rich oligonucleotides targeted to the two purine.pyrimidine strand biased regions near the DHFR transcription initiation site was developed. The stable triple helical structures formed by binding of the oligonucleotides to the native promoter double helix were dominated by G*G.C triplets, with interspersed C*C.G and A*A.T alignments. Mismatches between the oligonucleotide and the purine-rich strand of the target significantly destabilized third strand binding, and a G*A.T alignment was particularly unfavorable. Formation of a pur.pur.pyr triple helical structure results in a localized limitation of access to the native double helical DNA and produces sequence dependent conformational alterations extending several nucleotides beyond the triplex-duplex boundary. Although they differ only by the insertion of two A.T base pairs, the distal and proximal purine.pyrimidine regions can be targeted individually due to the high degree of sequence specificity of triple helical alignment. Triplex formation overlapping any of three consensus transcriptional regulatory elements and collectively covering 50% of the DHFR core promoter is now possible with this set of oligonucleotides.
Sequences located several kilobases both 5' and 3' of the stably transcribed portion of several genes hybridize to radio-labeled pure fragments of the alternating sequence poly (dG-dT) (dC-dA) ["poly(GT)"]. The genes include the ribosomal DNA of mouse, rat, and human, and also human glucose-6-phosphate dehydrogenase (G6PD) and mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT). HPRT has additional hybridizing sequences in introns. Fragments that include the hybridizing sequences and up to 300 bp of adjoining DNA show perfect runs of poly(GT) (greater than 30bp) in all but the human 5' region of rDNA, which shows a somewhat different alternating purine:pyrimidine sequence, poly(GTAT) (36bp). Within 150 bp of these sequences in various instances are found a number of other sequences reported to affect DNA conformation in model systems. Most marked is an enhancement of sequences matching at least 67% to the consensus binding sequence for topoisomerase II. Two to ten-fold less of such sequences were found in other sequenced portions of the nontranscribed spacer or in the transcribed portion of rDNA. The conservation of the locations of tracts of alternating purine:pyrimidine between evolutionarily diverse species is consistent with a possible functional role for these sequences.
The rat alpha1(I) collagen promoter contains a unique polypurine-polypyrimidine sequence between -141 and -200 upstream of the transcription start site. The polypurine sequence from -171 to -200 (C2) is on the coding strand and the adjacent polypurine sequence from -141 to -170 (C1) is on the non-coding strand. Earlier we demonstrated triplex formation with a polypurine 30 nt parallel triplex-forming oligonucleotide (TFO) corresponding to C1 and inhibition of transcriptional activity of the rat alpha1(I) collagen promoter. In the present work we have tested triplex-forming abilities of shorter (18 nt) purine and pyrimidine TFOs in parallel and antiparallel orientation to the C1 purine sequence. Our results show that purine antiparallel TFOs formed triplexes with the highest binding affinities, while pyrimidine oligodeoxyribonucleotides (ODNs) did not show appreciable binding. Phosphorothioate modification of purine TFOs did not significantly reduce binding affinity. We also demonstrate that preformed triplexes are quite stable when precipitated with ethanol and resuspended in water. Further analysis was carried out using two purine phosphorothioate antiparallel TFOs, 158 APS and 164 APS, designed to bind to the promoter region from -141 to -158 and -147 to -164, respectively, which were found to form triplexes even under physiological conditions. DNase I footprinting experiments showed the ability of these TFOs to protect target sequences in the promoter region; both purine sequences (C1 and C2) were protected in the case of 158 APS. Transfection experiments using preformed triplexes with a reporter plasmid containing the collagen promoter sequence showed significant inhibition of transcription when compared with a control phosphorothioate ODN. The effect of 164 APS was greater than that of 158 APS. These results indicate that this triplex strategy could be used in the down-regulation of collagen synthesis in cultured cells and offer the potential to control fibrosis in vivo.
Triplex-forming oligonucleotides (TFOs) have been shown to bind to target DNA sequences in several human gene promoters such as the c-myc oncogene, the epidermal growth factor receptor, and the dihydrofolate reductase genes. TFOs have been shown to inhibit transcription in vitro and gene expression in cell culture of the c-myc and other genes. The HER-2/neu oncogene, which is overexpressed in breast cancer and other human malignancies, contains a purine-rich sequence in its promoter, which is favorable for purine:purine:pyrimidine (R:R:Y) triplex formation. Although its function in the HER-2/neu promoter is unknown, this purine-rich site is homologous to a protein-binding sequence in the promoter of the epidermal growth factor receptor that is necessary for efficient transcription of this gene. We have shown that this sequence is a site for nuclear protein binding by incubation with a crude nuclear extract. We describe the formation of an interstrand triplex using a purine-rich oligonucleotide antiparallel to this purine-rich target sequence of the HER-2/neu promoter. Triplex formation by the oligonucleotide prevents protein binding to the target site in the HER-2/neu promoter in vitro. We have shown that this oligonucleotide is a potent and specific inhibitor of HER-2/neu transcription in an in vitro assay. The triplex target site contains a single pyrimidine base that does not conform to the R:R:Y triplex motif. In an attempt to abrogate the potentially destabilizing effects of this pyrimidine base on triplex formation, we have substituted an abasic linker for the pyrimidine residue in the triplex forming oligonucleotide. Triplex formation with the modified oligonucleotide appears to occur with approximately equivalent binding affinity. Triplex formation in the HER-2/neu oncogene promoter prevents transcription in vitro and may represent a future modality for specific inhibition of this gene in vivo.
It has been demonstrated that certain alternating purine and pyrimidine sequences may assume a left-handed Z-DNA conformation. In order to evaluate the possibility that Z-DNA is involved in the modulation of gene expression, we examined the ability of various synthetic DNA polymers to affect the transfection of herpes simplex virus thymidine kinase (HSVtk) gene in Ltk- cells using the DNA-calcium phosphate cotransfection technique. We found that potential Z-DNA forming polymers such as, poly(dG-m5dC) X poly(dG-m5dC) and poly(dG-dC) X poly(dG-dC), cotransfected with the tk gene decreased the level of Tk+ transformed colonies. In contrast, cotransfection of the tk gene with polymers which do not assume Z-conformation such as, poly(dG) X poly(dC) or poly(dA-dT) X poly(dA-dT) showed no effect on the number of colonies formed. About 50% inhibition of the Tk+ colony formation was obtained by 0.4 micrograms of poly(dG-m5dC) X poly(dG-m5dC), or by 2 micrograms of poly(dG-dC) X poly(dG-dC). DNA uptake into Ltk- cells was not significantly affected by any of these polymers. Approximately 20-42 base pairs (bp) long alternating dG-dC sequence linked at either the 5'-end or 3'-end of tk gene were cloned into plasmids. These recombinant plasmids, however, showed no remarkable effect upon the transfection of Ltk- cells. The DNAs of Tk+ colonies obtained by transfecting these recombinant plasmids were digested with BssH II and analyzed by Southern blotting. We demonstrated that the dG-dC sequences proximal to the tk gene were integrated into cellular DNA. All the presented results indicate that only larger polymers with the potential to assume a Z-DNA conformation may affect tk gene transfection either by inhibiting transcription or more probably by affecting the stable integration of the tk gene into the host chromosome.
In our previous works we have shown that the oligonucleotides 5'-GGGGAGGGGGAGG-3' and 5'-GGAGGGGGAGGGG-3' give very stable and specific triplexes with their target double stranded DNAs [Svinarchuk, F., Bertrand, J.-R. and Malvy, C. (1994) Nucleic Acids Res., 22, 3742-3747; Svinarchuk, F., Paoletti, J. and Malvy, C. (1995) J. Biol. Chem., 270, 14 068-14,071]. The target for the invariable part of these oligonucleotides, 5'-GGAGGGGGAGG-3', is found in a highly conserved 20 bp long purine/pyrimidine tract of the vpx gene of the SIV and HIV-2 viruses and could be a target for oligonucleotide directed antivirus therapy. Here were report on the ability of four purine oligonucleotides with different lengths (11-, 14-, 17- and 20-mer) to form triplexes with the purine/pyrimidine stretch of the vpx gene. Triplex formation was tested by joint dimethyl sulfate (DMS) footprint, gel-retardation assay, circular dichroism (CD) and UV-melting studies. Dimethyl sulfate footprint studies revealed the antiparallel orientation of the third strand to the purine strand of the Watson-Crick duplex. However, the protection of the guanines at the ends of the target sequence decreased as the length of the third strand oligonucleotide increased. Melting temperature studies provided profiles with only one transition for all of the triplexes. The melting temperatures of the triplexes were found to be the same as for the targeted duplex in the case of the 11- and 14-mer third strands while for the 17- and 20-mer third strands the melting temperature of the triplexes were correspondingly 4 and 8 degrees C higher than for the duplex. Heating and cooling melting curves were reversible for all of the tested triplexes except one with the 20-mer third strand oligonucleotide. Circular dichroism spectra showed the ability of the target DNA to adopt an A-like DNA conformation. Upon triplex formation the A-DNA form becomes even more pronounced. This effect depends on the length of the third strand oligonucleotide: the CD spectrum shows a 'classical' A-DNA shape with the 20-mer. This is not observed with the purine/pyrimidine stretch of the HIV-1 DNA which keeps a B-like spectrum even after triplex formation. We suggest, that an A-like duplex DNA is required for the formation of a stable DNA purine(purine-pyrimidine) triplex.
Two alternating purine-pyrimidine sequences of the d(TG)n.d(CA)n-type (170bp and 60 bp in length) lie upstream of the rat prolactin (rPRL) gene. Conformational studies of plasmids containing these sequences indicate that both form left-handed (Z) DNA, with transitions initiating at superhelical densities of -0.041 and -0.044 respectively. These alternating purine-pyrimidine (APP) sequences are hypersensitive to cleavage with S1 nuclease both at the boundaries and within these APP repeats, where there is a loss in APP alternation. We have investigated the function of one of these Z-DNA sequences in the regulation of rPRL transcription, by linking regions of the 5' flanking sequence of the rPRL gene to a reporter gene encoding chloramphenicol acetyltransferase (CAT), and transferring these plasmids into GH3 pituitary tumour cell lines. The major conclusion from these studies is that the 170bp repeat exerts a negative effect on the transcription of the rPRL gene, and also down-regulates the expression of the fusion gene pRSVcat when cloned 50bp upstream of the Rous sarcoma virus promoter. However, despite its proximity to an estrogen response element in prolactin, this sequence does not affect the responsiveness of the rPRL gene to estrogen.
The PHR1 gene of Saccharomyces cerevisiae encodes a photolyase which repairs specifically and exclusively pyrimidine dimers, the most frequent lesions induced in DNA by far-UV radiation. We have asked whether expression of PHR1 is modulated in response to UV-induced DNA damage and to DNA-damaging agents that induce lesions structurally dissimilar to pyrimidine dimers. Using a PHR1-lacZ fusion gene in which expression of beta-galactosidase is regulated by PHR1 5' regulatory elements, we found that exposure of cells to 254-nm light, 4-nitroquinoline-N-oxide, methyl methanesulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine induced synthesis of increased amounts of fusion protein. In contrast to these DNA-damaging agents, neither heat shock nor exposure to photoreactivating light elicited a response. Induction by far-UV radiation was evident both when the fusion gene was carried on a multicopy plasmid and when it replaced the endogenous chromosomal copy of PHR1, and it was accompanied by an increase in the steady-state concentration of PHR1-lacZ mRNA. Northern (RNA) blot analysis of PHR1 mRNA encoded by the chromosomal locus was consistent with either enhanced transcription of PHR1 after DNA damage or stabilization of the transcripts. Neither the intact PHR1 or RAD2 gene was required for induction. Comparison of the region of PHR1 implicated in regulation of its expression with other damage-inducible genes from yeast cells revealed a common conserved sequence that is present in the PHR1, RAD2, and RNR2 genes and is required for damage inducibility of the latter two genes. These sequences may constitute elements of a damage-responsive regulon in S. cerevisiae.
SNZ1, a member of a highly conserved gene family, was first identified through studies of proteins synthesized in stationary-phase yeast cells. There are three SNZ genes in Saccharomyces cerevisiae, each of which has another highly conserved gene, named SNO (SNZ proximal open reading frame), upstream. The DNA sequences and relative positions of SNZ and SNO genes have been phylogenetically conserved. This report details studies of the expression of the SNZ-SNO gene pairs under various conditions and phenotypic analysis of snz-sno mutants. An analysis of total RNA was used to determine that adjacent SNZ-SNO gene pairs are coregulated. SNZ2/3 and SNO2/3 mRNAs are induced prior to the diauxic shift and decrease in abundance during the postdiauxic phase, when SNZ1 and SNO1 are induced. In snz2 snz3 mutants, SNZ1 mRNA is induced prior to the diauxic shift, when SNZ2/3 mRNAs are normally induced. Under nitrogen-limiting conditions, SNZ1 mRNAs accumulate in tryptophan, adenine, and uracil auxotrophs but not in prototrophic strains, indicating that induction occurs in response to the limitation of specific nutrients. Strains carrying deletions in all SNZ-SNO gene pairs are viable, but snz1 and sno1 mutants are sensitive to 6-azauracil (6-AU), an inhibitor of purine and pyrimidine biosynthetic enzymes, and methylene blue, a producer of singlet oxygen. The conservation of sequence and chromosomal position, the coregulation and pattern of expression of SNZ1 and SNO1 genes, and the sensitivity of snz1 and sno1 mutants to 6-AU support the hypothesis that the associated proteins are part of an ancient response to nutrient limitation.
The GAL1 and GAL10 genes, separated by 680 base pairs and divergently transcribed on chromosome 2 of Saccharomyces cerevisiae, were separately fused to the lacZ gene of Escherichia coli so that beta-galactosidase synthesis in S. cerevisiae reflected GAL1 and GAL10 promoter function. Analysis of two sets of deletions defined a 75-base-pair sequence, located ca. midway between the transcription initiation regions of GAL1 and GAL10, that mediates GAL4-dependent induction of both genes. Deletion of various parts of this sequence (called the GAL upstream activating sequence or UASG) reduced GAL1 and GAL10 induction about equally. Sequences in the GAL10-proximal half of UASG in some sequence contexts functioned independently of sequences in the GAL1-proximal half of UASG. A 33-base-pair deletion of the GAL10-proximal half of UASG drastically reduced induction. Deletions between UASG and the GAL1 TATA box caused beta-galactosidase to be synthesized at an unexpectedly high basal level, that is, in the absence of galactose and GAL4 product. Some of these mutations also reduced the repression caused by glucose.
Eukaryotic organisms contain mitochondria, organelles capable of producing large amounts of ATP by oxidative phosphorylation. Each cell contains many mitochondria with many copies of mitochondrial DNA in each organelle. The mitochondrial DNA encodes a small but functionally critical portion of the oxidative phosphorylation machinery, a few other species-specific proteins, and the rRNA and tRNA used for the translation of these transcripts. Because the microenvironment of the mitochondrion is unique, mitochondrial genes may be subject to different selectional pressures than those affecting nuclear genes.
From an analysis of the mitochondrial genomes of a wide range of eukaryotic species we show that there are three simple rules for the pyrimidine and purine abundances in mitochondrial DNA transcripts. Mitochondrial membrane protein transcripts are pyrimidine rich, rRNA transcripts are purine-rich and the soluble protein transcripts are purine-rich. The transitions between pyrimidine and purine-rich regions of the genomes are rapid and are easily visible on a pyrimidine-purine walk graph. These rules are followed, with few exceptions, independent of which strand encodes the gene. Despite the robustness of these rules across a diverse set of species, the magnitude of the differences between the pyrimidine and purine content is fairly small. Typically, the mitochondrial membrane protein transcripts have a pyrimidine richness of 56%, the rRNA transcripts are 55% purine, and the soluble protein transcripts are only 53% purine.
The pyrimidine richness of mitochondrial-encoded membrane protein transcripts is partly driven by U nucleotides in the second codon position in all species, which yields hydrophobic amino acids. The purine-richness of soluble protein transcripts is mainly driven by A nucleotides in the first codon position. The purine-richness of rRNA is also due to an abundance of A nucleotides. Possible mechanisms as to how these trends are maintained in mtDNA genomes of such diverse ancestry, size and variability of A-T richness are discussed.
We have carried out a systematic investigation of the efficiency of misincorporation by Avian Myeloblastosis Virus reverse transcriptase with all possible combinations of dNTP substrate, template nucleotide, and the nucleotide at the 3' terminus of the primer. A series of synthetic oligonucleotide primers were annealed to single stranded M13 DNA templates, and a single dNTP was misincorporated at the primer 3' end using AMV reverse transcriptase. The proportion and pattern of misincorporation and incorporation in all 64 situations was assayed using [5'-32p] labelled primers, and the products were separated on denaturing polyacrylamide gels. Correct incorporations occurred more readily than misincorporations. The efficiency of misincorporation depended on the individual primer, but, comparing primers, a clear dependence on the template nucleotide was observed for the preferential misincorporation of different dNTPs. The exact combination of template and dNTP was important; although purine:pyrimidine (dNTP substrate:template nucleotide) and pyrimidine:purine misincorporations occurred comparatively readily, some pyrimidine:pyrimidine and purine:purine reactions were equally efficient and yet others were never seen to occur. Some misincorporations were facilitated by subsequent correct incorporations, but despite this our results suggest that the level of misincorporation is limited by the rate of reaction and enzyme inactivation rather than by exonuclease activity.
In addition to the well-known internal promoter elements of tRNA genes, 5' flanking sequences can also influence the efficiency of transcription by Saccharomyces cerevisiae extracts in vitro. A consensus sequence of yeast tRNA genes in the vicinity of the transcriptional start site can be derived. To determine whether the activity of this region can be attributed to particular sequence features we studied in vitro mutants of the start site region. We found that the start site can be shifted, but only to a limited extent, by moving the conserved sequence element. We found that both a pyrimidine-purine motif (with transcription initiating at the purine) and a small T:A base pair block upstream are important for efficient transcription in vitro. Thus the sequence surrounding the start site of transcription of the yeast tRNA(Leu3) gene does play a role in determining transcription efficiency and fixing the precise site of initiation by RNA polymerase III.
Triplex-forming oligonucleotides (TFOs) bind DNA in a sequence-specific manner at polypurine/polypyrimidine sites and mediate targeted genome modification. Triplexes are formed by either pyrimidine TFOs, which bind parallel to the purine strand of the duplex (pyrimidine, parallel motif), or purine TFOs, which bind in an anti-parallel orientation (purine, anti-parallel motif). Both purine and pyrimidine TFOs, when linked to psoralen, have been shown to direct psoralen adduct formation in cells, leading to mutagenesis or recombination. However, only purine TFOs have been shown to mediate genome modification without the need for a targeted DNA-adduct. In this work, we report the ability of a series of pyrimidine TFOs, with selected chemical modifications, to induce repair and recombination in two distinct episomal targets in mammalian cells in the absence of any DNA-reactive conjugate. We find that TFOs containing N3′→P5′ phosphoramidate (amidate), 5-(1-propynyl)-2′-deoxyuridine (pdU), 2′-O-methyl-ribose (2′-O-Me), 2′-O-(2-aminoethyl)-ribose, or 2′-O, 4′-C-methylene bridged or locked nucleic acid (LNA)-modified nucleotides show substantially increased formation of non-covalent triplexes under physiological conditions compared with unmodified DNA TFOs. However, of these modified TFOs, only the amidate and pdU-modified TFOs mediate induced recombination in cells and stimulate repair in cell extracts, at levels comparable to those seen with purine TFOs in similar assays. These results show that amidate and pdU-modified TFOs can be used as reagents to stimulate site-specific gene targeting without the need for conjugation to DNA-reactive molecules. By demonstrating the potential for induced repair and recombination with appropriately modified pyrimidine TFOs, this work expands the options available for triplex-mediated gene targeting.
DNA bending is believed to facilitate the initial recognition of the mismatched base for repair. The repair efficiencies are dependent on both the mismatch type and neighboring nucleotide sequence. We have studied bending of several DNA duplexes containing canonical matches: A:T, G:C, various mismatches: A:A, A:C, G:A, G:G, G:T, C:C, C:T, T:T, and a bis-abasic site: X:X. Free energy profiles were generated for DNA bending using umbrella sampling. The highest energetic cost associated with DNA bending is observed for canonical matches while bending free energies are lower in the presence of mismatches, with the lowest value for the abasic site. In all of the sequences, DNA duplexes bend towards the major groove with widening of the minor groove. For homoduplexes, DNA bending is observed to occur via smooth deformations, whereas for heteroduplexes, kinks are observed at the mismatch site during strong bending. In general, pyrimidine:pyrimidine mismatches are the most destabilizing, while purine:purine mismatches lead to intermediate destabilization and purine:pyrimidine mismatches are the least destabilizing. The ease of bending is partially correlated with the binding affinity of MutS to the mismatch pairs and subsequent repair efficiencies, indicating that intrinsic DNA bending propensities are a key factor of mismatch recognition.
Umbrella sampling; free energy; mismatch recognition; DNA repair
We constructed a series of deletions in the 5' noncoding region of the Saccharomyces cerevisiae GAL7 gene, fused them to the Escherichia coli gene lacZ, and introduced them into yeasts by using a multicopy vector. We then studied the effect of the deletions on beta-galactosidase synthesis directed by the gene fusions in media with various carbon sources. This analysis identified a TATA box and two upstream activating sequences as necessary elements for galactose-controlled GAL7 transcription. Two upstream activating sequences exhibiting 71% homology with each other were located 255 and 168 base pairs, respectively, upstream of the GAL7 transcription start point. Each sequence consists of 21 base pairs, displaying an approximate rotational symmetry with a core consensus sequence of GAA--AGCTGCTTC--CGCG. At least one of the two sequences is required for galactose induction and also for glucose repression of the GAL7'-lac'Z gene. Analysis with host regulatory mutants delta gal14 and delta gal180 suggests that these sequences are the site at which the GAL4 product exerts its action to activate the GAL7 gene. We also observed that a deletion lacking both upstream activation sequences allowed the gene fusion to be expressed in the absence of galactose at about 10% of the fully induced level of the intact fusion. This constitutive expression depended on the presence of the TATA box of GAL7 in cis but not on a functional GAL4 gene. The level of the uncontrolled expression was decreased by increasing the distance between the TATA box and the pBR322 sequence in the vector plasmid.
(Pyrimidine)n . (purine)n DNAs of repeating sequences form a distinctive complex on lowering the pH below 6. Previously this complex was thought to be tetra-stranded. The present work is inconsistent with this view, and four lines of evidence show that the complex consists of a triplex together with a poly d(purine) possessing secondary structure. Formula: (see text). (a) S1 nuclease digestion leads to degradation of 50% of the poly d(purine) content of the pH 5-induced complex. (b) Buoyant density studies demonstrate that there is no interaction between the triplex and added free poly d(purine) and also that the complex formed from duplex DNA contained poly d(purine) which is free to form a triplex on addition of an appropriate poly d(pyrimidine) in the correct stoichiometry. (c) The hyperchromic shifts of the triplex and poly d(purine), upon melting, are mutually independent. (d) The circular dichroism spectrum of the complex is simply the weighted average of a triplex together with a free poly d(purine). The triplexes have tm's approximately 20 degrees higher than the corresponding duplexes under comparable conditions and they are extremely resistant to various deoxyribonucleases; properties which may prove useful for their isolation from natural sources.
Yeast mutants assigned to the pet complementation group G104 were found to lack alpha-ketoglutarate dehydrogenase activity as a result of mutations in the dihydrolipoyl transsuccinylase (KE2) component of the complex. The nuclear gene KGD2, coding for yeast KE2, was cloned by transformation of E250/U6, a G104 mutant, with a yeast genomic library. Analysis of the KGD2 sequence revealed an open reading frame encoding a protein with a molecular weight of 52,375 and 42% identities to the KE2 component of Escherichia coli alpha-ketoglutarate dehydrogenase complex. Disruption of the chromosomal copy of KGD2 in a respiratory-competent haploid yeast strain elicited a growth phenotype similar to that of G104 mutants and abolished the ability to mitochondria to catalyze the reduction of NAD+ by alpha-ketoglutarate. The expression of KGD2 was transcriptionally regulated by glucose. Northern (RNA) analysis of poly(A)+ RNA indicated the existence of two KGD2 transcripts differing in length by 150 nucleotides. The concentrations of both RNAs were at least 10 times lower in glucose (repressed)- than in galactose (derepressed)-grown cells. Different 5'-flanking regions of KGD2 were fused to the lacZ gene of E. coli in episomal plasmids, and the resultant constructs were tested for expression of beta-galactosidase in wild-type yeast cells and in hap2 and hap3 mutants. Results of the lacZ fusion assays indicated that transcription of KGD2 is activated by the HAP2 and HAP3 proteins. The regulated expression of KGD2 was found to depend on sequences that map to a region 244 to 484 nucleotides upstream of the structural gene. This region contains two short sequence elements that differ by one nucleotide from the consensus core (5'-TN[A/G]TTGGT-3') that has been proposed to be essential for binding of the HAP activation complex. These data together with earlier reports on the regulation of the KGD1 and LPD1 genes for the alpha-ketoglutarate and dihydrolipoyl dehydrogenases indicate that all three enzyme components of the complex are catabolite repressed and subject to positive regulation by the HAP2 and HAP3 proteins.
In vitro assembly of an intermolecular purine*purine.pyrimidine triple helix requires the presence of a divalent cation. The relationships between cation coordination and triplex assembly were investigated, and we have obtained new evidence for at least three functionally distinct potential modes of divalent cation coordination. (i) The positive influence of the divalent cation on the affinity of the third strand for its specific target correlates with affinity of the cation for coordination to phosphate. (ii) Once assembled, the integrity of the triple helical structure remains dependent upon its divalent cation component. A mode of heterocyclic coordination/chelation is favorable to triplex formation by decreasing the relative tendency for efflux of integral cations from within the triple helical structure. (iii) There is also a detrimental mode of base coordination through which a divalent cation may actively antagonize triplex assembly, even in the presence of other supportive divalent cations. These results demonstrate the considerable impact of the cationic component, and suggest ways in which the triple helical association might be positively or negatively modulated.
Poly(pyrimidine) . poly(purine) tracts have been discovered in the 5'-flanking regions of many eucaryotic genes. They may be involved in the regulation of expression since they can be mapped to the nuclease-sensitive sites of active chromatin. We have found that poly(pyrimidine) . poly(purine) DNAs which contain 5-methylcytosine (e.g. poly[d(Tm5C)] . poly[d(GA)]) will form a triplex at a pH below 8. In contrast, the unmethylated analogue, poly[d(TC)] . poly[d(GA)] only forms a triplex at pHs below 6. Synthetic DNAs containing repeating trinucleotides and poly[d(Um5C)] . poly[d(GA)] behave in a similar manner. Thus the stability of a triplex can be controlled by methylation of cytosine. This suggests a model for the regulation of expression based upon specific triplex formation on the 5'-side of eucaryotic genes.
Nucleoside hydrolases catalyze the cleavage of N-glycosidic bonds in nucleosides, yielding ribose and the respective bases. While nucleoside hydrolase activity has not been detected in mammalian cells, many protozoan parasites rely on nucleoside hydrolase activity for salvage of purines and/or pyrimidines from their hosts. In contrast, uridine phosphorylase is the key enzyme of pyrimidine salvage in mammalian hosts and many other organisms. We show here that the open reading frame (ORF) YDR400w of Saccharomyces cerevisiae carries the gene encoding uridine hydrolase (URH1). Disruption of this gene in a conditionally pyrimidine-auxotrophic S. cerevisiae strain, which is also deficient in uridine kinase (urk1), leads to the inability of the mutant to utilize uridine as the sole source of pyrimidines. Protein extracts of strains overexpressing YDR400w show increased hydrolase activity only with uridine and cytidine, but no activity with inosine, adenosine, guanosine, and thymidine as substrates, demonstrating that ORF YDR400w encodes a uridine-cytidine N-ribohydrolase. Expression of a homologous cDNA from a protozoan parasite (Crithidia fasciculata) in a ura3 urk1 urh1 mutant is sufficient to restore growth on uridine. Growth can also be restored by expression of a human uridine phosphorylase cDNA. Yeast strains expressing protozoan N-ribohydrolases or host phosphorylases could therefore become useful tools in drug screens for specific inhibitors.
The upstream activating sequence of the adjacent and divergently transcribed GAL1 and GAL10 genes of Saccharomyces cerevisiae (UASG) contains at least three distinct classes of overlapping transcriptional control sites. The transcription activator GAL4 binds to four related sites in UASG and induces expression of GAL1 and GAL10 when galactose is available. We showed that UASG contains two additional positive control sites, designated GAL4/galactose-independent activating elements (GAEs), which reside at positions adjacent to or overlapping the GAL4-binding sites. When separated from neighboring sequences in UASG, the GAEs activate transcription independently of GAL4 with no requirement for galactose. In the intact GAL1-GAL10 divergent promoter region, their activity is ordinarily repressed by multiple negative control elements, the GAL operators. When galactose is available, GAL4 overcomes the activity of the GAL operators, while the putative GAE-binding proteins stay repressed. Combined, these results imply that distinct activators (GAL4 and GAE proteins) bound at adjacent or overlapping sites in UASG are differentially regulated by putative repressor proteins simultaneously bound at adjacent GAL operators. We surmise that GAE1 and GAE2 may have a physiological function other than regulation of galactose catabolism per se and discuss three hypotheses to account for their presence in UASG.
Similar to tRNA genes and the VAI gene, the Alu family repeats are transcribed by RNA polymerase III and contain a split intragenic promoter. Results of our previous studies have shown that when the anterior, box A-containing promoter element (5'-Pu-Pu-Py-N-N-Pu-Pu-Py-G-G-3' in which Pu is any purine, Py is any pyrimidine, and N is any nucleotide) of a human Alu family repeat is deleted, the remaining box B-containing promoter element (5'-G-A/T-T-C-Pu-A-N-N-C-3') is still capable of directing weak transcriptional initiation at approximately 70 base pairs (bp) upstream from the box B sequence. This is different from the tRNA genes in which the box A-containing promoter element plays the major role in the positioning of the transcriptional initiation site(s). To account for this difference, we first carried out competition experiments in which we show that the posterior element of the Alu repeat competes with the VAI gene effectively for the transcription factor C in HeLa cell extracts. We then constructed a series of contraction and expansion mutants of the Alu repeat promoter in which the spacing between boxes A and B was systematically varied by molecular cloning. In vitro transcription of these clones in HeLa cell extracts was analyzed by RNA gel electrophoresis and primer extension mapping. We show that when the box A and box B promoter sequences are separated by 47 to 298 bp, the transcriptional initiation sites remain 4 to 5 bp upstream from box A. However, this positioning function by the box A-containing promoter element was lost when the spacing was shortened to only 26 bp or increased to longer than 600 bp. Instead, transcriptional initiation occurred approximately 70 bp upstream from box B, similar to that in the clones containing only the box B promoter element. All the mutant clones were transcribed less efficiently than was the wild type. An increase in the distance between boxes A and B also activated a second box A-like element within the Alu family repeat. We compare these results with the results of tRNA gene studies. We also discuss this comparison in terms of the positioning function of the split class III promoter elements and the evolutionary conservation of the spacing between the two promoter elements for optimum transcriptional efficiency.
The transcriptional activator protein GCN4 is responsible for increased transcription of more than 30 different amino acid biosynthetic genes in response to starvation for a single amino acid. This induction depends on increased expression of GCN4 at the translational level. We show that starvation for purines also stimulates GCN4 translation by the same mechanism that operates in amino acid-starved cells, being dependent on short upstream open reading frames in the GCN4 mRNA leader, the phosphorylation site in the alpha subunit of eukaryotic translation initiation factor 2 (eIF-2 alpha), the protein kinase GCN2, and translational activators of GCN4 encoded by GCN1 and GCN3. Biochemical experiments show that eIF-2 alpha is phosphorylated in response to purine starvation and that this reaction is completely dependent on GCN2. As expected, derepression of GCN4 in purine-starved cells leads to a substantial increase in HIS4 expression, one of the targets of GCN4 transcriptional activation. gcn mutants that are defective for derepression of amino acid biosynthetic enzymes also exhibit sensitivity to inhibitors of purine biosynthesis, suggesting that derepression of GCN4 is required for maximal expression of one or more purine biosynthetic genes under conditions of purine limitation. Analysis of mRNAs produced from the ADE4, ADE5,7, ADE8, and ADE1 genes indicates that GCN4 stimulates the expression of these genes under conditions of histidine starvation, and it appeared that ADE8 mRNA was also derepressed by GCN4 in purine-starved cells. Our results indicate that the general control response is more global than was previously imagined in terms of the type of nutrient starvation that elicits derepression of GCN4 as well as the range of target genes that depend on GCN4 for transcriptional activation.