A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of ammalian 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 framents distributed along a given genomic region. Terminal differentiation of HL-60 was achieved with retinoic acid and dimethylformamide, as described (17).Transfection. Plasmid pAWTSV (=9 kb), a kind gift of Cesare Vesco (Institute of Cell Biology, Rome), carries the whole simian virus 40 (SV40) genome inserted in the BamHI site of pAT153 (18). Six 10-cm tissue culture plates, containing about 106 COS-1 cells each, were transfected with 10 pg of pAWTSV by the calcium phosphate precipitation technique. After 10 hr of incubation in calcium phosphate solution, cells were extensively washed and fresh medium was added, containing 10 nCi of [14C]thymidine per ml. After 18 hr of incubation, BrdUrd (100 uM final concentration) and[3H]deoxycytidine (1 jAM final concentration) were added.After 1 min of incubation, cells were killed by addition of sodium azide and DNA was extracted as described below.Extrction and Purification of Newly Syntez DNA.Total DNA was extracted, denatured, and size-fractionated by sedimentation through neutral sucrose gradients as described (15).In the experiment involving transfection of plasmid pAW-TSV, DNA (700 /4 final volume) was fractionated on four 5-20%6 (wt/vol) linear sucrose gradients (5 ml each) for 210 min at 200C in a Beckman SW55Ti rotor at 55 krpm; 24 fractions of 200 j4 were collected.In the experiment with synchronized HL-60 cells, DNA (2 ml final volume) was fractionated on eight 5-30% sucrose Abbreviations: DHFR, dihydrofolate reductase; SV40, simian virus 40. tTo whom reprint requests should be addressed. 7119The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of doublehelical 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.Oligonucleotides can be designed to bind to double-stranded DNA at oligopurine⅐oligopyrimidine sites where they form a local triple-helical structure (see refs. 1 and 2 for reviews). This provides a general approach to the sequence-specific recognition and targeting of double-stranded DNA for both basic research and therapeutic applications. However, the description of triplex-mediated effects in cell cultures is still rare. Inhibition of gene expression by triplex-forming oligonucleotides has been demonstrated on plasmid targets transiently transfected into living cells (3-7). When a triplex was preformed with the target in vitro and then the complex was transfected into mammalian cells, inhibition of gene expression was observed. We have previously shown that transcription of the gene coding for the ␣-subunit of the interleukin 2 receptor was inhibited when cells were first transfected with a reporter plasmid in the presence of a psoralenoligonucleotide conjugate and then irradiated (3), or when transfected cells were incubated with an acridineoligonucleotide conjugate without any irradiation (8, 9). Mutations have been detected on plasmid vectors after irradiation of transfected cells in the presence of psoralenoligonucleotide conjugates (10). Even in the absence of irradiation a low level of mutation was detected and was attributed to transcription-coupled repair (11). Location of the mutation sites was as it was expected on the basis of triplex formation. Detection of the DNA mutations is a very sensitive method to demonstrate triplex formation, but it does not allow quantitative analysis of the amount of triplex formed within cells. Using dimethyl sulfate footprinting ...

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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.

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.

tHcy and plasma vitamin B6 are long-term independent risk factors for coronary and cerebrovascular events.

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