Site-directed inhibition of DNA replication by triple helix formation.

Sequence-specific DNA recognition can be achieved by the use of triplex-forming molecules, namely, oligonucleotides (TFO) and peptide nucleic acids (PNAs). They have been used to regulate transcription or induce genomic DNA modifications at a selected site in cells and, recently, in vivo. We have determined the conditions under which a triplex structure can inhibit DNA replication in cells. An oligopyrimidine.oligopurine sequence suitable for triplex formation was inserted in a plasmid on both sides of the SV40 origin of replication. This insert-containing plasmid was replicated in COS-1 cells together with the parent plasmid, and the ratio between the corresponding replicated DNAs was quantitated. Selective inhibition of replication of the insert-containing plasmid can be ascribed to ligand binding to the oligopyrimidine.oligopurine sequence. Inhibition of DNA replication was observed using triplex-forming molecules that induce either covalent binding at the double-stranded target sequence (with TFO-psoralen conjugate and irradiation) or noncovalent triplex formation after strand displacement (with bis-PNA). In contrast, in the absence of covalent cross-linking, TFOs (which have been shown to arrest transcription elongation) did not act on replication. These results open new perspectives for future design and use of specific inhibitors of intracellular DNA information processing.

Downregulation of c-Ki-ras promoter activity by triplex-forming oligonucleotides endogenously generated in human 293 cells.

Exogenous triplex-forming oligodeoxynucleotides (TFO) have the capacity to modulate in vivo the expression of individual genes. As the administration of TFO to cells is not without problems, we analyzed the possibility of generating them directly in the cell, using specific expression vectors. We constructed three vectors, mU6-GA, mU6-CA, and mU6-CT, that direct the synthesis in human 293 cells of 76-mer CU, GU, and AG motif TFO (rTFO) potentially capable of binding to a critical poly (R x Y) sequence contained in the promoter of the Ki-ras proto-oncogene. The ability of the CU, GU, and AG motif rTFO to interact with the double helix of the c-Ki-ras target was investigated in vitro by footprinting and band-shift experiments, using both synthetic and endogenously synthesized oligoribonucleotides. The human 293 cells were transfected with DNA mixtures containing a plasmid, which bears the reporter chloramphenicol acetyltransferase (CAT) gene downstream from the c-Ki-ras promoter (pKRS-413), as well as an rTFO-generating vector (mU6-GA, mU6-CA, or mU6-CT). As control, the cells were transfected with DNA mixtures containing vector mU6-C1 or mU6-C2. These generated transcripts unable to form triple helices with the poly (R x Y) sequence of the c-Ki-ras promoter. Intracellular synthesis of the 76-mer CU, GU, and AG rTFO by mU6-GA, mU6-CA, and mU6-CT was checked by Northern blot hybridization. Through beta-gal and CAT ELISA immunoassays, we found that the 293 cells transfected with either mU6-GA, mU6-CA, or mU6-CT showed a significant inhibition of CAT expression compared with cells transfected with control plasmids mU6-C1 or mU6-C2. The results of five separate transient transfection experiments showed that endogenous GU and AG rTFO, generated by mU6-CA and mU6-CT, produce, respectively, 40% (+/- 4% SE) and 47% (+/- 8% SE) CAT inhibition, whereas CU rTFO, generated by mU6-GA, produces 38% (+/- 7% SE) CAT inhibition. In conclusion, this study suggests that it is possible to downregulate the expression of an individual gene through the use of recombinant vectors encoding the information for the intracellular synthesis of short triplex-forming RNA strands.

Accessibility of nuclear DNA to triplex-forming oligonucleotides: the integrated HIV-1 provirus as a target.

The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming oligonucleotides within cell nuclei. An oligonucleotide-psoralen conjugate targeted to the polypurine tract (PPT) of the HIV-1 proviral sequence was used as a tool to convert the noncovalent triple-helical complex into a covalent lesion on genomic DNA after UV irradiation of cells. Triplex-derived adducts were analyzed using two different methods. The photo-induced psoralen cross-link prevented cleavage of the target sequence by DraI restriction endonuclease, and the sequence-specific inhibition of cleavage was revealed and quantitated by Southern blot analysis. A quantitative analysis of cross-linking efficiency was also carried out by a competitive PCR-based assay. These two approaches allowed us to demonstrate that a triplex-forming oligonucleotide can recognize and bind specifically to a 15-bp sequence within the chromatin structure of cell nuclei.

High-resolution mapping of the origin of DNA replication in the hamster dihydrofolate reductase gene domain by competitive PCR.

By the use of a highly sensitive mapping procedure allowing the identification of the start sites of DNA replication in single-copy genomic regions of untreated, exponentially growing cultured cells (M. Giacca, L. Zentilin, P. Norio, S. Diviacco, D. Dimitrova, G. Contreas, G. Biamonti, G. Perini, F. Weighardt, S. Riva, and A. Falaschi, Proc. Natl. Acad. Sci. USA 91:7119-7123, 1994), the pattern of DNA replication of the Chinese hamster dihydrofolate reductase (DHFR) gene domain was investigated. The method entails the purification of short stretches of nascent DNA issuing from DNA replication origin regions and quantification, within this sample, of the abundance of different adjacent segments by competitive PCR. Distribution of marker abundance peaks around the site from which newly synthesized DNA had emanated. The results obtained by analysis of the genomic region downstream of the DHFR single-copy gene in asynchronous cultures of hamster CHO K1 cells are consistent with the presence of a single start site for DNA replication, located approximately 17 kb downstream of the gene. This site is coincident with the one detected by other studies using different techniques in CHO cell lines containing an amplified DHFR gene domain.

Fine mapping of a replication origin of human DNA.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.

Searching for replication origins in mammalian DNA.

The attempts at identifying precise replication origins (ori) in mammalian DNA have been pursued mainly through physico-chemical and biochemical approaches, in view of the essential failure of the search for autonomously replicating sequences in cultured cells. These approaches involve the mapping of short stretches of nascent DNA, the identification of the regions where either leading or lagging strands switch polarity, or the localization of replication intermediates by two-dimensional gel electrophoresis. Due to the complexity of animal cell genomes, most of these studies have been performed on amplified domains and with the use of synchronization procedures. The results obtained have been controversial. In order to avoid the use of experimental procedures potentially affecting the physiological mechanism of DNA replication, we have developed a method for the localization of ori in single-copy loci in exponentially growing cells. This method entails the absolute quantification of the abundance of selected DNA fragments along a genomic region within samples of newly synthesized DNA by competitive polymerase chain reaction (PCR); the latter is immune to all the uncontrollable variables which severely affect the reproducibility of conventional PCR. The application of this method to SV40 ori-driven plasmid replication precisely identifies the known ori localization. Using the same approach, we have mapped an ori for bi-directional DNA replication in a 13.7-kb locus of human chromosome 19 encoding lamin B2.

A novel procedure for quantitative polymerase chain reaction by coamplification of competitive templates.

A method is described for the absolute quantification by polymerase chain reaction (PCR) of nucleic acids present in low abundance. The method entails the addition to the sample of competitor DNA molecules that share the same sequence as the amplified target (including primer recognition sites), except for a 20-bp insertion in the middle, which allows easy resolution by gel electrophoresis (competitive PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial rate of targets, rendering the reaction independent of the number of amplification cycles. An easy and reliable method for the construction and quantification of competitive templates obtained as recombinant PCR products was developed. The technique was used for the absolute quantification of human genomic DNA with primers from a single copy, subtelomeric region of chromosome 19.

A human DNA replication origin: localization and transcriptional characterization.

A single-copy 13.7 kb human DNA region (L30E) located on Ch. 19 p13.3 contains an origin of DNA replication in myeloid HL-60 cells. The origin was localized, by means of quantitative PCR within approximately 3000 bp, in a highly transcribed region containing at least two closely spaced genes with the same polarity of transcription, one encoding lamin B2 and the other an unidentified protein. The origin region overlaps an undermethylated "CpG island" at the 5'-end of the second transcription unit. A binding site (CACGTG) for basic helix-loop-helix (bHLH) DNA binding proteins such as USF/MLTF or MYC-MAX was located by DNase I footprinting analysis in the promoter of the second gene. DMSO differentiation of HL-60 cells, that completely shuts off replication, also drastically reduces the transcription of L30E region. On the other hand such treatment does not modify the methylation pattern of the CpG island and does not abolish the DNase I protection of the bHLH binding site.

A protein target site in an early replicated human DNA sequence: a highly conserved binding motif.

We have previously reported that a human nuclear factor, probably corresponding to the USF/MLTF protein [1,2], is able to bind specifically to a DNA sequence present in DNA replicated at the onset of S-phase [3]. Here we demonstrate that the same factor binds also to several other similar sequences, present in eukaryotic and viral genomes. Mutations or methylation in a CpG dinucleotide, central in the palindromic binding site, completely abolish binding. Furthermore, we present evidence for the existence of at least two other nuclear proteins in human cells with the same DNA binding specificity. The data presented suggest a strong evolutionary conservation, among distantly related organisms, of the binding motif, which is probably the target of a number of nuclear factors that share the same DNA binding specificity albeit in the context of different functions.